Detection, isolation and uses of renalase (monoamine oxidase c)

ABSTRACT

The present invention provides for the identification, isolation and uses of mammalian Monoamine Oxidase C (MAO-C), also known as renalase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is entitled to priority pursuant to 35 U.S.C. §119(e)to U.S. Provisional Patent Application No. 60/554,552, which was filedon Mar. 19, 2004 and to U.S. Provisional Patent Application No.60/615,452, which was filed on Oct. 1, 2004, both which are incorporatedherein in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported in part by funds obtained from the U.S.Government (National Institutes of Health Grant Number K08 DK 0291702),and the U.S. Government may therefore have certain rights in theinvention.

BACKGROUND OF THE INVENTION

The regulation of fluid and electrolyte metabolism is a major functionof the kidney. Blood enters the kidney through the glomerulus, whichfilters out cells and proteins and generates, through a process calledglomerular filtration, a fluid with an ionic composition identical tothat of plasma. The glomerular filtrate then travels through a series ofdistinct tubular segments, which progressively modify its volume andionic composition. A large number of factors are known to regulateglomerular filtration including physical forces, local and circulatinghormones (Brenner et al. 1976). Similarly, renal tubular reabsorptionand secretion are modified by rather complex regulatory processes.

The kidney also serves as an endocrine organ, as it is the main sourceof erythropoietin, a major determinant of red cell mass that is requiredfor amplification and terminal differentiation of erythroid progenitorsand precursors (Line et al., 1985; Jacobs et al., 1985). In addition,the kidney appears to be the most important site for renin release. Afall in blood flow, increased sympathetic stimulation, or a decrease insodium delivery to the distal tubules can stimulate the release ofrenin, an enzyme that cleaves angiotensinogen to angiotensin I. Therenin-angiotensin system is a key regulator of fluid and electrolytemetabolism, blood pressure, and cardiac function.

Among these many functions carried out by the kidney, the importance ofunderstanding the kidney endocrine function is underscored by thediscovery of erythropoietin. There is evidence to suggest that thekidney has complex endocrine functions beyond secretion of renin anderythropoietin. The identification of previously unknownproteins/hormones that are secreted by the kidney will not only providea more complete understanding of renal physiology but may alsosignificantly improve the way we treat patients with end-stage renaldiseases (ESRD).

Patients who develop end-stage renal disease are either treated withrenal replacement therapy, such as peritoneal or hemodialysis, or givena renal transplantation. Current renal replacement therapy such ashemodialysis for patients with end-stage renal disease has been the onlysuccessful long-term ex vivo organ substitution therapy to date. Despitethe success of dialysis at prolonging life, the morbidity and mortalityassociated with this therapy are undesirably high, and most patientssuffer from a poor quality of life (Humes et al., 1995; Wolfe et al.1999). For example, these patients have increased prevalence ofhypertension, cardiovascular diseases such as asymptomatic leftventricular dysfunction, chronic congestive heart failure andatherosclerosis, contributing the most common cause of death among them.While the reasons for this are not entirely clear, it is generallybelieved that the procedure fails to replicate important functions ofthe natural organ. The procedure uses an extracorporeal “artificialkidney” to remove excess water and soluble wastes from the blood butdoes not replicate the important absorptive, metabolic, endocrine, andimmunological functions of the natural organ.

It is well documented that patients with ESRD are at significantlyhigher risk for developing cardiovascular disease, a risk that appearsto be correlated with increased oxidative stress (Oberge et al. 2004)and heightened sympathetic tone (Koomans et al., 2004; Joles et al.,2004). Despite the fact that various proteins/hormones have beenimplicated in ESRD, very few factors involved in ESRD have beenidentified and characterized. Nevertheless, the identification of suchfactors is crucial in the development of diagnostics and therapeuticsfor treatment of ESRD and vascular diseases associated orproteins/hormones secreted by the kidney. Thus, there is long-felt needfor the identification and characterization of factors associated withESRD.

Monoamine oxidase (MAO) is a flavin-adenosine-dinucleotide(FAD)-containing enzyme which converts biogenic amines to theircorresponding aldehydes. MAO is present as two isoforms (MAO-A (SEQ IDNO: 11) and MAO-B (SEQ ID NO: 13)), which are separate gene products,that exhibit more than 70% sequence identity and distinct butoverlapping substrate specificities in the catabolism ofneurotransmitters, such as dopamine, serotonin and norepinephrine (2,3).Both MAO-A and MAO-B are implicated in a large number of neurologicaldisorders and are targets for drugs against Parkinson's disease anddepression (4). Mammalian MAOs are bound to the outer mitochondrialmembrane and have a FAD molecule covalently bound to the protein via an8α-thioether linkage to a cysteinyl residue (5). They are expressed inboth a tissue-dependent and an age-dependent manner and have been thesubject of extensive clinical and pharmacological studies.

MAO-A and MAO-B are anchored through the carboxyl terminus to the outermitochondrial membrane (Binda et al., 2002). They have overlappingsubstrate specificity, catabolize neurotransmitters such as epinephrine,norepinephrine, serotonin and dopamine, and are specifically inhibitedby pargyline and clorgyline. Polyamine oxidase (PAO), the other knownFAD-containing oxidase, is an intracellular oxidase that metabolizesspermine and spermidine, and regulates cell growth (Jalkanen et al.,2001). The crystal structure of human MAO-B has been solved at aresolution of 3.0 A, and reveals a dimer with the FAD cofactorcovalently bound to a cysteine side chain (Cys-397) (Binda et al.,2002). MAO-A and MAO-B are coded by adjoining, but separate, genes onthe X chromosome, that exhibit over 70% sequence identity and distinctbut overlapping substrate specificities in the catabolism ofneurotransmitters.

MAO-A and MAO-B differ in tissue distribution, structure and substratespecificity. MAO-A has higher affinity for serotonin, octopamine,adrenaline, and noradrenaline; whereas the natural substrates for MAO-Bare phenylethylamine and tyramine. Dopamine is thought to be oxidized byboth isoforms. MAO-A and MAO-B are widely distributed in several organsincluding brain (A. M. Cesura and A. Pletscher, Prog. Drug Research1992, 38, 171-297). Brain MAO-B activity appears to increase with age.This increase has been attributed to the gliosis associated with aging(C. J. Fowler et al., J. Neural. Transm. 1980, 49, 1-20). Additionally,MAO-B activity is significantly higher in the brains of patients withAlzheimer's disease (P. Dostert et al., Biochem. Pharmacol. 1989, 38,555-561) and it has been found to be highly expressed in astrocytesaround senile plaques (Saura et al., Neuroscience 1994, 70, 755-774). Inthis context, since oxidative deamination of primary monoamines by MAOproduces NH₃, aldehydes and H₂O₂, agents with established or potentialtoxicity, it is suggested that there is a rationale for the use ofselective MAO-B inhibitors for the treatment of dementia and Parkinson'sdisease. Inhibition of MAO-B causes a reduction in the enzymaticinactivation of dopamine and thus prolongation of the availability ofthe neurotransmitter in dopaminergic neurons. The degeneration processesassociated with age and Alzheimer's and Parkinson's diseases may also beattributed to oxidative stress due to increased MAO activity andconsequent increased formation of H₂O₂ by MAO-B. Therefore, MAO-Binhibitors may act by both reducing the formation of oxygen radicals andelevating the levels of monoamines in the brain.

Given the implication of MAO-B in the neurological disorders mentionedabove, there is considerable interest to obtain potent and selectiveinhibitors that would permit control over this enzymatic activity. Thepharmacology of some known MAO-B inhibitors is for example discussed byD. Bentue-Ferrer et al. in CNS Drugs 1996, 6, 217-236. Whereas a majorlimitation of irreversible and non-selective MAO inhibitor activity isthe need to observe dietary precautions due to the risk of inducing ahypertensive crisis when dietary tyramine is ingested, as well as thepotential for interactions with other medications (D. M. Gardner et al.,J. Clin. Psychiatry 1996, 57, 99-104), these adverse events are of lessconcern with reversible and selective MAO inhibitors, in particular ofMAO-B.

By inhibiting MAO activity, MAO inhibitors can regulate the level ofmonoamines and their neurotransmitter release in different brain regionsand in the body (including dopamine, norepinephrine, and serotonin).Thus, MAO inhibitors can affect the modulation of neuroendocrinefunction, respiration, mood, motor control and function, focus andattention, concentration, memory and cognition, and the mechanisms ofsubstance abuse. Inhibitors of MAO have been demonstrated to haveeffects on attention, cognition, appetite, substance abuse, memory,cardiovascular function, extrapyramidal function, pain andgastrointestinal motility and function. The distribution of MAO in thebrain is widespread and includes the basal ganglia, cerebral cortex,limbic system, and mid and hind-brain nuclei. In the peripheral tissue,the distribution includes muscle, the gastrointestinal tract, thecardiovascular system, autonomic ganglia, the liver, and the endocrinicsystem.

MAO inhibition by other inhibitors have been shown to increase monoaminecontent in the brain and body. Regulation of monoamine levels in thebody have been shown to be effective in numerous disease statesincluding depression, anxiety, stress disorders, diseases associatedwith memory function, neuroendocrine problems, cardiac dysfunction,gastrointestinal disturbances, eating disorders, hypertension,Parkinson's disease, memory disturbances, and withdrawal symptoms.

It has been suggested that cigarette smoke may have irreversibleinhibitory effect towards monoamine oxidase (MAO). Boulton et al.,“Biogenic Amine Adducts, Monoamine Oxidase Inhibitors, and Smoking,”Lancet, 1(8577): 114-155 (Jan. 16, 1988), reported that theMAO-inhibiting properties of cigarette smoke may help to explain theprotective action of smoking against Parkinson's disease and alsoobserved that patients with mental disorders who smoke heavily do notexperience unusual rates of smoking-induced disorders. It was suggestedthat smoking, as an MAO inhibitor, may protect against dopaminergicneurotoxicity that leads to Parkinson's disease and that theMAO-inhibiting properties of smoking may result in an anti-depressiveeffect in mental patients.

SUMMARY OF THE INVENTION

Prior to the invention, MAO-A and MAO-B are the only two monoamineoxidases identified in human. While attempting to identify andcharacterize novel secretory proteins in human using human genomicsdatabase, the present inventors identified a new monoamine oxidase. Thepresent invention discloses the identification and characterization of asecretory form of monoamine oxidase that metabolizes biogenic monoaminessuch as norepinephrine, dopamine and epinephrine. Since the newlyidentified monoamine oxidase is the third enzyme identified thatmetabolize monoamines in human, the present inventors designated it bythe name ‘MAO-C’. Alternatively, because it is a protein secreted by thekidney, it is also termed ‘renalase.’ Because MAO-C, or renalase, is anew member of the monoamine oxidase family, the enzyme is also expectedto play an important role in the brain as do MAO-A and MAO-B.

The present invention relates a nucleic acid encoding a novel mammalianmonoamine oxidase (MAO) termed renalase (SEQ ID NO: 2), a novel proteininvolved in regulating catecholamines. The nucleic acid sequence ofhuman renalase is 27.7% homologous to that of human MAO-A (SEQ ID NO:10) and 38.2% homologous to that of MAO-B (SEQ ID NO: 12). Renalase has13% and 12% identity at the amino acid level to MAO-A and MAO-B,respectively. It also has a distinct substrate specificity and inhibitorprofile to that of MAO-A and MAO-B, indicating that it represents abrand new class of unique FAD-containing monoamine oxidases.

The human renalase gene resides on chromosome 10, contains 9 exons andspans about 300 Kb. The human renalase gene encodes a 342-amino acidprotein that contains an amino-terminal signal sequence, followed by aflavin-adenosine-dinucleotide (FAD)-containing domain and an aminooxidase domain. Tissue Northern blotting studies demonstrated robustexpression of renalase in kidney with much lower levels in all othertissues analyzed. In situ hybridization demonstrated the high level ofexpression of renalase in proximal and distal tubules.

Renalase was highly expressed in in vitro transcription and translationexperiments. Human renalase cDNA is translated to produce a protein witha molecular mass of approximately 38-kDa, which is in agreement with thepredicted protein size. Western blotting studies using conditionedmedium from transfected HEK293 cells indicates that renalase is asecreted protein. Renalase is present in the plasma at a concentrationof 5-10 mg/l in healthy individual.

End-Stage Renal Disease (ESRD) is associated with elevated catecholaminelevels, which in turn leads to a myriad of conditions, diseases anddisorders, including, for example, asymptomatic left ventriculardysfunction, chronic congestive heart failure and atherosclerosis. Theseconditions, diseases and disorders are a common cause of death amongESRD patients.

Renalase metabolizes catecholamines in the following rank orders:dopamine>epinephrine>norepinephrine. Ranalase is virtually undetectablein patients with ESRD. Thus, the loss or reduced levels of renalase inESRD patients is at least in part responsible for elevated plasmacatecholamine levels, which leads to increased cardiovascular disease,which is a common cause of death among ESRD patients.

In addition, the correlation between renalase levels and renal functionmake renalase an ideal candidate for a diagnostic marker for renaldisease, especially for acute tubular necrosis, a common occurrence inthe Intensive Care Unit setting. The biological significance of renalaseand its potential clinical relevance are further discussed herein.

The present invention provides a novel FAD-dependent amine oxidase thatis secreted into the blood by the kidney. It metabolizes circulatingcatecholamines and inter alia is a potent regulator of blood pressureand heart rate. Furthermore, its reduced presence in the plasma ofpatients with ESRD suggests a causal link to the heightened sympathetictone and increased cardiovascular risks that are well documented in thispatient population. The identification of renalase is not only animportant step in development of a more detailed understanding ofcardiovascular physiology, but also an important step in the quest forproviding optimal treatment for patients with kidney disease and/orheart disease and their related complications.

The invention includes an isolated nucleic acid molecule encoding apolypeptide, wherein the nucleic acid molecule shares at least about orgreater than about 40% sequence identity with the nucleic acid sequenceof SEQ ID NO: 1.

The invention includes an isolated nucleic acid molecule encoding apolypeptide, wherein the nucleic acid molecule shares at least about orgreater than about 40% sequence identity with the nucleic acid sequenceof nucleic acid residues 24 to 1049 of SEQ ID NO: 1.

In one aspect, the nucleic acid further comprises a nucleic acidencoding a tag polypeptide covalently linked thereto.

In another aspect, the nucleic acid further comprises a nucleic acidspecifying a promoter/regulatory sequence operably linked thereto.

In yet another aspect, the invention includes a vector comprising anisolated nucleic acid encoding a polypeptide, wherein the nucleic acidshares at least about or greater than about 40% identity with SEQ ID NO:1.

In a further aspect, the invention includes a recombinant cellcomprising an isolated nucleic acid encoding a polypeptide, wherein thenucleic acid shares at least about or greater than about 40% identitywith SEQ ID NO: 1.

The invention includes an isolated nucleic acid complementary to anisolated nucleic acid encoding a polypeptide, or a fragment thereof, thecomplementary nucleic acid being in an antisense orientation.

In one aspect, a nucleic acid of the present invention shares at leastabout or greater than about 40% identity with a nucleic acidcomplementary with a nucleic acid having the sequence of a humanrenalase (SEQ ID NO: 1).

The invention includes an isolated mammalian renalase peptide,polypeptide or protein.

The invention includes an isolated mammalian polypeptide, wherein thepolypeptide comprises an amino acid sequence having at least about orgreater than about 15% identity with a polypeptide having the amino acidsequence of SEQ ID NO: 2.

The invention also includes an isolated polypeptide, wherein thepolypeptide comprises an amino acid sequence having at least about orgreater than about 15% identity with a polypeptide having the amino acidsequence of amino acid residues 24 to 342 of SEQ ID NO: 2.

The invention includes an antibody that specifically binds with amammalian renalase, or a fragment thereof. The antibody can be apolyclonal antibody, a monoclonal antibody, or a synthetic antibody.

The invention includes a composition comprising an isolated nucleic acidencoding a polypeptide, wherein the nucleic acid shares at least aboutor greater than about 40% identity with SEQ ID NO: 1, and apharmaceutically-acceptable carrier.

The invention includes a composition comprising an isolated mammalianrenalase polypeptide and a pharmaceutically-acceptable carrier.

The invention includes a method of identifying a compound that reducesor inhabits expression of human renalase in a cell. The method comprisescontacting a cell in which renalase is expressed with a compound andcomparing the level of expression of human renalase in the cellcontacted with the compound with the level of expression of humanrenalase in an otherwise identical cell, wherein a lower level ofexpression of human renalase in the cell contacted with the compoundcompared with the level of expression of human renalase in the otherwiseidentical cell not contacted with the compound, is an indication thatthe compound inhibits expression of human renalase in the cell. In oneaspect, the invention includes a compound identified by this method,wherein such a compound is an antagonist of human renalase.

The invention also includes a method of identifying a compound thatenhances or increases expression of human renalase in a cell. The methodcomprises contacting a cell in which renalase is expressed with acompound and comparing the level of expression of human renalase in thecell contacted with the compound with the level of expression of humanrenalase in an otherwise identical cell, wherein a higher level ofexpression of human renalase in the cell contacted with the compoundcompared with the level of expression of human renalase in the otherwiseidentical cell not contacted with the compound, is an indication thatthe compound enhances or increases expression of human renalase in thecell. In one aspect, the invention includes a compound identified bythis method, wherein such a compound is an agonist of human renalase.

The invention includes a method of treating a condition, disorder ordisease mediated by expression of a human renalase. The method comprisesadministering to a human patient afflicted with a condition, disorder ordisease mediated by expression of a human renalase, a human renalaseexpression-inhibiting or human renalase expression-reducing amount of arenalase inhibitor, thereby treating a condition, disorder or diseasemediated by expression of a human renalase.

The compositions and methods of the present invention may be used totreat any condition, disorder or disease associated with the vascular,cardiac, renal, neural and/or endocrine systems of an organism,including humans. In one aspect, the condition, disorder or disease isselected from the group consisting of ESRD, chronic kidney disease,hypertension, cardiovascular diseases such as asymptomatic leftventricular dysfunction, chronic congestive heart failure, cardiacrhythm disturbances, and atherosclerosis.

In yet another aspect, the renalase inhibitor comprises an isolatednucleic acid complementary to an isolated nucleic acid encoding a humanrenalase, or a fragment thereof, the complementary nucleic acid being inan antisense orientation.

The invention includes a method of treating hypertension in a mammal,comprising administering renalase, thereby treating hypertension in themammal.

Also contemplated is a method of treatment of conditions, disorders ordiseases of the central nervous system (CNS) including withoutlimitation dementia, Alzheimer's disease, schizophrenia, psychosis,depression, headaches, migraine headache or a tension headache andepilepsy; and treatment and/or prevention of CNS disorders such as majordepressive disorders including bipolar depression, unipolar depression,single or recurrent major depressive episodes with or without psychoticfeatures, catatonic features, melancholic features, atypical features orpostpartum onset, the treatment of anxiety and the treatment of panicdisorders. Other mood disorders encompassed within the term majordepressive disorders include dysthymic disorder with early or late onsetand with or without atypical features, neurotic depression, posttraumatic stress disorders and social phobia; dementia of Alzheimer'stype, with early or late onset, disorders induced by alcohol,amphetamines, cocaine, hallucinogens, inhalants, opiods, phecyclidine,sedatives, hypnotics, anxiolytics and other substances.

The invention further includes a method of identifying a human patientafflicted with a disease, disorder or condition associated with alteredexpression of renalase. The method comprises detecting the level ofrenalase expression in a human and comparing the level of expression ofrenalase in the human with the level of expression of renalase in anormal human not afflicted with a disease, disorder or conditionassociated with altered expression of renalase, thereby detecting ahuman patient afflicted with a disease, disorder or condition associatedwith altered expression of renalase.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiment(s) which are presentlypreferred. It should be understood, however, that invention is notlimited to the precise arrangements and instrumentalities shown. In thedrawings:

FIG. 1A is a Northern blot analysis of human tissue using the MGC12474clone as a probe.

FIG. 1B depicts putative structural motifs detected in renalase: FAD:flavin adenine dinucleotide, SP: signal peptide.

FIG. 1C is deduced amino sequence of human renalase in comparison withMAO-A.

FIG. 1D is a Western blot analysis of rat tissue using a renalasepolycolonal antibody.

FIG. 2A shows the cDNA and deduced amino acid sequence of humanrenalase. Amino acid residues 1-16 enclosed by a box are believed tocode for a signal peptide.

FIG. 2B depicts the genomic structure of renalase.

FIG. 2C is an alignment between human MAO-A, MAO-B and MAO-C.

FIGS. 3A-3D show subcellular localization of renalase in the humankidney and heart.

FIG. 3A is an in situ hybridization analysis of human kidney; Leftpanel: antisense probe, magnification of 200×, open arrows label theglomerulus, closed arrows indicate proximal tubules; right panel: senseprobe control magnification of 100×.

FIG. 3B is an in situ hybridization analysis of human heart; Left panel:antisense probe, magnification of 200×, open arrows label blood vessels,closed arrows indicate ventricular myocytes; right panel: sense probecontrol.

FIG. 3C is an image of immunolocalization in human kidney; Left panel:anti-renalase antibody, magnification of 630×, closed arrows indicateproximal tubules; right panel: preimmune serum.

FIG. 3D is an image of immunolocalization in human heart; Left panel:anti-renalase antibody, magnification of 630×, open arrows label bloodvessels, closed arrows indicate ventricular myocytes; right panel:preimmune serum.

FIGS. 4A through 4C show the expression of a rat renalase-HA-taggedfusion protein in HEK293 cells.

FIG. 4A depicts the construction of expression TAP fragment of renalase.

FIG. 4B is an image depicting the generation and purification ofGST-renalase fusion protein. The proteins (100 μg) from crude bacteriallysate and purified GST-renalase (10 μg) were separated on a 10%SDS-polyacrylamide gel, stained with Coomassie blue. Lane A: crudebacterial lysate; lane B: purified renalase protein.

FIG. 4C shows the determination of renalase activity. PurifiedGST-renalase fusion protein (10 μg for each reaction) was used for eachassay. The amino oxidase activity is expressed in arbitrary fluorescenceunits/10 μg of protein after 30 min incubation with dopamine ornorepinephrine (2 mM final concentration). Column 1: Control protein(GST) with dopamine and norepinephrine; Column 2: GST-renalase withnorepinephrine; Column 3: GST-renalase with dopamine.

FIGS. 5A through 5B show that renalase is a secreted protein.

FIG. 5A is an image depicting the detection of renalase in culturemedium of HEK293 cells transiently transfected with renalase cDNA.

FIG. 5B is an imaging depicting the Western blot analysis of humanplasma using an anti-renalase antibody, normal refers to individualswith normal renal function, control is recombinant renalase, ESRDrepresents patients with end-stage renal disease receiving hemodialysis.

FIGS. 6A through 6C show enzymatic activity and function of renalase.

In FIG. 6A, 10 μg of GST-renalase fusion protein was used for eachassay; amine oxidase activity is expressed in arbitrary fluorescenceunits/10 μg of protein; substrates (2 mM) are incubated for 30 minincubation.

In FIG. 6B, as in 6A; 1 μM chlorgyline and 1 μM pargyline was used.

FIG. 6C is an image of affinity purified human renalase. Protein wasisolated from human urine using the anti-renalase antibody; lane 1:human urine, lane 2: control with secondary antibody alone.

FIGS. 7A through 7G depict the hemodynamic effects of renalase; arrowsdenotes the timing of renalase injection, time scale is in minutes,heart rate is measured in beats per minute (bpm) and systolic anddiastolic arterial blood pressures are expressed in millimeters ofmercury (mm Hg).

FIG. 7A shows cardiac response before and after an IV bolus injection of4 μg/g body weight; arrow denotes the timing of renalase injection;Vp=left ventricular pressure; HR=heart rate; dP/dt=rate of change inleft ventricular pressure, a measure of cardiac contractility.

FIG. 7B is the pressure-volume curve before and after renalaseinjection.

FIG. 7C is an enlargement of a portion of FIG. 7A (see Arrow). HR: heartrate, Vp: left ventricular pressure.

FIG. 7D is the renalase dose-response curve on cardiac contractility.

FIG. 7E is the renalase dose-response curve on mean arterial pressure.

FIG. 7F is the time course of renalase activity on plasma epinephrine.

FIG. 7G is the time course of renalase activity on plasmanorepinephrine.

DETAILED DESCRIPTION OF THE INVENTION

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

The data disclosed herein do not exclude receptor interaction and theydemonstrate that renalase plays a role in, inter alia, degradingcirculating catecholamines such as norepinephrine, dopamine, andepinephrine, which are important mediators for the function of thekidney, heart and blood vessels. As described more fully elsewhereherein, renalase therefore also plays a role in acting as a hormone inthat it regulates the function of other tissues and/or organs. This mayalso include the nervous system, as its functions are also mediated inpart by catecholamines. Thus, renalase can be provided, for example, topatients who do not produce sufficient amounts of renalase.Identification of renalase has important implications in the developmentof therapeutics and diagnostics for, among other things, end-stage renaldiseases (ESRD) to treat/prevent hypertension, cardiovascular diseasessuch as asymptomatic left ventricular dysfunction, chronic congestiveheart failure and atherosclerosis.

Similar to erythropoietin (EPO), a recombinant protein widely used totreat patients with anemia, renalase is a secreted protein and isexpressed in the kidney. Another striking feature of renalase is that,like EPO, it is virtually non-detectable in patients with ESRD, whereasrenalase is expressed in the blood of healthy individuals atconcentration of about 5-10 mg/L.

The data disclosed herein demonstrate the existence of a novel enzymethat metabolizes catecholamines. In addition to expanding the paradigmof MAO, this new finding has significant clinical implications for, forexample, ESRD patients. Strategies whereby renalase levels arereplenished to counteract excessive levels of renalase substrates (i.e.,catecholamines) are logical approaches to treating patients with ESRD.Furthermore, any other disease caused by excessive levels ofcatecholamines, regardless of the status of renal function, could betreated through the addition of either supplemental or replenishingamounts of renalase.

Alternatively, renalase can be used as a drug target in order to raisecirculatory catecholamine levels in patients with decreased sympathetictone, and thus improve the outcome of certain cardiovascularcomplications. Renalase may also allow for the design of drugs morespecific for MAO-A or MAO-B, as current MAO inhibitors may unknowinglyalso target renalase, which may result in unfavorable side-effects.

The identification and characterization of renalase provides a frameworkfor further study of renalase and its role in the pathophysiology ofcardiovascular diseases such as chronic heart failure (CHF), myocardialinfarction (MI), cardiac rhythm disturbances as these diseases may beprecipitated by sudden emotional stress which increases sympatheticstimulation (Wittstein et al., (2005) Neurohumoral features ofmyocardial stunning due to sudden emotional stress, New England Journalof Medicine, 352, 539-548). Perhaps more importantly, like MAO-A and -B,renalase may provide a potentially useful target for modulatingsympathetic activity in human.

In addition to its potential therapeutic role, renalase can be used as adiagnostic marker for acute renal failure (i.e. acute tubular necrosis,or ATN, an ischemic condition in the kidney). As described above,patients without a properly functioning kidney possess lower levels ofrenalase.

Also included in the invention are methods of diagnosing susceptibilityto cardiovascular, heart, kidney and mental related conditions,disorders and diseases based on the measurement of gene expression andenzyme activity of renalase. For example, cardiovascular conditions,disorders and diseases such as hypertension, asymptomatic leftventricular dysfunction, chronic congestive heart failure, myocardialinfarction, cardiac rhythm disturbance, and atherosclerosis; mentalconditions, disorders and diseases such as depression and anxiety; andheart conditions, disorders and diseases, such as pulmonaryhypertension, can all be diagnosed, evaluated and monitored bydetermining renalase gene expression levels, renalase protein levels,and/or renalase enzyme activity. For example, reduced expression of therenalase gene would be a diagnostic marker for a disorder associatedwith an increased sympathetic output.

The compositions and methods of the present invention can be used totreat, prevent, reduce or ameliorate hypertension, including systolichypertension, isolated systolic hypertension and diabetic hypertension.Moreover, the same benefit is anticipated for the more rare hypertensivedisorder, pulmonary hypertension. Pulmonary hypertension is a rare bloodvessel disorder of the lung in which the pressure in the pulmonaryartery (the blood vessel that leads from the heart to the lungs) risesabove normal levels and may become life threatening. The similarity indevelopment of elevated blood pressure in the pulmonary bed with theincrease in systemic blood pressure in diabetic hypertension and inisolated systolic hypertension suggests similar mechanisms are involved.

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “adjacent” is used to refer to nucleotidesequences which are directly attached to one another, having nointervening nucleotides. By way of example, the pentanucleotide5′-AAAAA-3′ is adjacent the trinucleotide 5′-TTT-3′ when the two areconnected thus: 5′-AAAAATTT-3′ or 5′-TTTAAAAA-3′, but not when the twoare connected thus: 5′-AAAAACTTT-3′.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

As used herein, to “alleviate” a disease, disorder or condition meansreducing the severity of one or more symptoms of the disease, disorderor condition. This can include, but is not limited to, increasing thelevel of renalase expressed in a cell or tissue (e.g., smooth musclecell, lung tissue, an artery), reducing or increasing the level ofrenalase in a patient, compared with the level of renalase in thepatient prior to or in the absence of the method of treatment, and thelike.

“Antisense” refers particularly to the nucleic acid sequence of thenon-coding strand of a double stranded DNA molecule encoding a protein,or to a sequence which is substantially homologous to the non-codingstrand. As defined herein, an antisense sequence is complementary to thesequence of a double stranded DNA molecule encoding a protein. It is notnecessary that the antisense sequence be complementary solely to thecoding portion of the coding strand of the DNA molecule. The antisensesequence may be complementary to regulatory sequences specified on thecoding strand of a DNA molecule encoding a protein, which regulatorysequences control expression of the coding sequences.

By the term “applicator” as the term is used herein, is meant any deviceincluding, but not limited to, a hypodermic syringe, a pipette, abronchoscope, a nebulizer, and the like, for administering the renalasenucleic acid, protein, and/or composition of the invention to a mammal.

“Biological sample,” as that term is used herein, means a sampleobtained from an animal that can be used to assess the level ofexpression of a renalase, the level of renalase protein present, orboth. Such a sample includes, but is not limited to, a blood vessel(e.g., carotid artery, aorta, and the like) sample, a lung tissuesample, and a SMC sample.

By “complementary to a portion or all of the nucleic acid encodingrenalase” is meant a sequence of nucleic acid which does not encode arenalase protein. Rather, the sequence which is being expressed in thecells is identical to the non-coding strand of the nucleic acid encodinga renalase protein and thus, does not encode renalase protein.

The terms “complementary” and “antisense” as used herein, are notentirely synonymous. “Antisense” refers particularly to the nucleic acidsequence of the non-coding strand of a double stranded DNA moleculeencoding a protein, or to a sequence which is substantially homologousto the non-coding strand.

“Complementary” as used herein refers to the broad concept of subunitsequence complementarity between two nucleic acids, e.g., two DNAmolecules. When a nucleotide position in both of the molecules isoccupied by nucleotides normally capable of base pairing with eachother, then the nucleic acids are considered to be complementary to eachother at this position. Thus, two nucleic acids are complementary toeach other when a substantial number (at least 50%) of correspondingpositions in each of the molecules are occupied by nucleotides whichnormally base pair with each other (e.g., A:T and G:C nucleotide pairs).As defined herein, an antisense sequence is complementary to thesequence of a double stranded DNA molecule encoding a protein. It is notnecessary that the antisense sequence be complementary solely to thecoding portion of the coding strand of the DNA molecule. The antisensesequence may be complementary to regulatory sequences specified on thecoding strand of a DNA molecule encoding a protein, which regulatorysequences control expression of the coding sequences.

A “coding region” of a gene consists of the nucleotide residues of thecoding strand of the gene and the nucleotides of the non-coding strandof the gene which are homologous with or complementary to, respectively,the coding region of an mRNA molecule which is produced by transcriptionof the gene.

A “coding region” of an mRNA molecule also consists of the nucleotideresidues of the mRNA molecule which are matched with an anticodon regionof a transfer RNA molecule during translation of the mRNA molecule orwhich encode a stop codon. The coding region may thus include nucleotideresidues corresponding to amino acid residues which are not present inthe mature protein encoded by the mRNA molecule (e.g., amino acidresidues in a protein export signal sequence).

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., retroviruses, adenoviruses, and adeno-associatedviruses) that incorporate the recombinant polynucleotide.

The term “replication defective” as used herein relative to a viral genetherapy vector of the invention means the viral vector cannotindependently further replicate and package its genome. For example,when a cell of a subject is infected with rAAV virions, the heterologousgene is expressed in the infected cells, however, due to the fact thatthe infected cells lack AAV rep and cap genes and accessory functiongenes, the rAAV is not able to replicate.

As used herein, a “retroviral transfer vector” refers to an expressionvector that comprises a nucleotide sequence that encodes a transgene andfurther comprises nucleotide sequences necessary for packaging of thevector. Preferably, the retroviral transfer vector also comprises thenecessary sequences for expressing the transgene in cells.

As used herein, “packaging system” refers to a set of viral constructscomprising genes that encode viral proteins involved in packaging arecombinant virus. Typically, the constructs of the packaging systemwill ultimately be incorporated into a packaging cell.

As used herein, a “second generation” lentiviral vector system refers toa lentiviral packaging system that lacks functional accessory genes,such as one from which the accessory genes, vif, vpr, vpu and nef, havebeen deleted or inactivated. See, e.g., Zufferey et al., 1997, Nat.Biotechnol. 15:871-875.

As used herein, a “third generation” lentiviral vector system refers toa lentiviral packaging system that has the characteristics of a secondgeneration vector system, and further lacks a functional tat gene, suchas one from which the tat gene has been deleted or inactivated.Typically, the gene encoding rev is provided on a separate expressionconstruct. See, e.g., Dull et al., 1998, J. Virol. 72(11):8463-8471.

As used herein, “pseudotyped” refers to the replacement of a nativeenvelope protein with a heterologous or functionally modified envelopeprotein.

As used herein, “ex vivo administration” refers to a process whereprimary cells are taken from a subject, a vector is administered to thecells to produce transduced, infected or transfected recombinant cellsand the recombinant cells are readministered to the same or a differentsubject.

A first region of an oligonucleotide “flanks” a second region of theoligonucleotide if the two regions are adjacent one another or if thetwo regions are separated by no more than about 1000 nucleotideresidues, and preferably no more than about 100 nucleotide residues.

As used herein, the term “fragment” as applied to a nucleic acidsequence or nucleic acid molecule refers to a segment or portion of areference full length nucleic acid sequence or molecule, wherein thefragment is less than the full length of the reference nucleic acidsequence or molecule. An example of a fragment nucleic acid sequence ormolecule is a segment or portion of the full length renalase cDNAsequence or molecule, respectively. Another example of a fragmentnucleic acid sequence or molecule is a segment or portion of a fulllength genomic renalase DNA sequence or molecule, respectively. Afragment may be any length that is less than the full length of thenatural or native cDNA or gene. Examples of fragments include nucleicacids of at least about 20 nucleotides in length, at least about 50nucleotides, at least about 50 to about 100 nucleotides, at least about100 to about 200 nucleotides, at least about 200 nucleotides to about300 nucleotides, at least about 300 to about 350, at least about 350nucleotides to about 500 nucleotides, at least about 500 to about 600,at least about 600 nucleotides to about 650 nucleotides, at least about650 to about 800, or at least 800 to about 1000 nucleotides in length.

As used herein, the term “fragment” as applied to an amino acid sequenceor amino acid molecule refers to a segment or portion of a referencefull length amino acid sequence or molecule, wherein the fragment isless than the full length of the reference amino acid sequence ormolecule. An example of a fragment amino acid sequence or molecule is asegment or portion of a polypeptide encoded by the full length renalasecDNA sequence or molecule, respectively. Another example of a fragmentamino acid sequence or molecule is a segment or portion of a polypeptideencoded by a full length genomic renalase DNA sequence or molecule,respectively. A fragment may be any length of a polypeptide that is lessthan the full length polypeptide encoded by a natural or native cDNA orgene. Examples of fragments include amino acids of at least about 20amino acids in length, or at least about 30 amino acids, or at leastabout 40, or at least about 50, or at least about 60, or at least about70, or at least about 80, or at least about 90, or at least about 100,or at least about 110, or at least about 120, or at least about 130, orat least about 130, or at least about 140, or at least about 150, or atleast about 160, or at least about 170, or at least about 180, or atleast about 190, or at least about 200, or at least about 210, or atleast about 220, or at least about 230, or at least about 240, or atleast about 250, or at least about 260, or at least about 270, or atleast about 280, or at least about 290, or at least about 300, or atleast about 310, or at least about 320, or at least about 330, or atleast about 340 amino acids in length.

A “genomic DNA” is a DNA strand which has a nucleotide sequencehomologous with a gene. By way of example, both a fragment of achromosome and a cDNA derived by reverse transcription of a mammalianmRNA are genomic DNAs.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′ATTGCC5′ and 3′TATGGC share 50%homology.

As used herein, “homology” is used synonymously with “identity.” Inaddition, when the terms “homology” or “identity” are used herein torefer to the nucleic acids and proteins, it should be construed to beapplied to homology or identity at both the nucleic acid and the aminoacid sequence levels. A first oligonucleotide anneals with a secondoligonucleotide with “high stringency” or “under high stringencyconditions” if the two oligonucleotides anneal under conditions wherebyonly oligonucleotides which are at least about 60%, more preferably atleast about 65%, even more preferably at least about 70%, yet morepreferably at least about 80%, and preferably at least about 90% or,more preferably, at least about 95% complementary anneal with oneanother. The stringency of conditions used to anneal twooligonucleotides is a function of, among other factors, temperature,ionic strength of the annealing medium, the incubation period, thelength of the oligonucleotides, the G-C content of the oligonucleotides,and the expected degree of non-homology between the twooligonucleotides, if known. Methods of adjusting the stringency ofannealing conditions are known (see, e.g., Sambrook et al., 1989, In:Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York).

The determination of percent identity between two nucleotide or aminoacid sequences can be accomplished using a mathematical algorithm. Forexample, a mathematical algorithm useful for comparing two sequences isthe algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl.Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into theNBLAST and XBLAST programs of Altschul et al. (1990, J. Mol. Biol.215:403-410), and can be accessed, for example, at the BLAST site of theNational Center for Biotechnology Information (NCBI) world wide web siteat the National Library of Medicine (NLM) at the National Institutes ofHealth (NIH). BLAST nucleotide searches can be performed with the NBLASTprogram (designated “blastn” at the NCBI web site), using the followingparameters: gap penalty=5; gap extension penalty=2; mismatch penalty=3;match reward=1; expectation value 10.0; and word size=11 to obtainnucleotide sequences homologous to a nucleic acid described herein.BLAST protein searches can be performed with the XBLAST program(designated “blastn” at the NCBI web site) or the NCBI “blastp” program,using the following parameters: expectation value 10.0, BLOSUM62 scoringmatrix to obtain amino acid sequences homologous to a protein moleculedescribed herein.

To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997, Nucleic Acids Res.25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used toperform an iterated search which detects distant relationships betweenmolecules (id.) and relationships between molecules which share a commonpattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used as available on the website of theNational Center for Biotechnology Information of the National Library ofMedicine at the National Institutes of Health.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a polypeptideof the invention. Such natural allelic variations can typically resultin 1-5% variance in the nucleotide sequence of a given gene. Alternativealleles can be identified by sequencing the gene of interest in a numberof different individuals. This can be readily carried out by usinghybridization probes to identify the same genetic locus in a variety ofindividuals. Any and all such nucleotide variations and resulting aminoacid polymorphisms or variations that are the result of natural allelicvariation and that do not alter the functional activity are intended tobe within the scope of the invention.

Moreover, nucleic acid molecules encoding proteins of the invention fromother species (homologs), which have a nucleotide sequence which differsfrom that of the mouse proteins described herein are within the scope ofthe invention. Nucleic acid molecules corresponding to natural allelicvariants and homologs of a cDNA of the invention can be isolated basedon their identity to mouse nucleic acid molecules using the mouse cDNAs,or a portion thereof, as a hybridization probe according to standardhybridization techniques under stringent hybridization conditions. Forexample, a homolog of a nucleic acid encoding a rat renalase protein ofthe invention can be isolated based on its hybridization with a nucleicacid molecule encoding all or part of rat and/or human renalase underhigh stringency conditions.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins. The term thereforeincludes, for example, a recombinant DNA which is incorporated into avector, into an autonomously replicating plasmid or virus, or into thegenomic DNA of a prokaryote or eukaryote, or which exists as a separatemolecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCRor restriction enzyme digestion) independent of other sequences. It alsoincludes a recombinant DNA which is part of a hybrid gene encodingadditional polypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

By describing two polynucleotides as “operably linked” is meant that asingle-stranded or double-stranded nucleic acid moiety comprises the twopolynucleotides arranged within the nucleic acid moiety in such a mannerthat at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized upon the other. By wayof example, a promoter operably linked to the coding region of a gene isable to promote transcription of the coding region. Preferably, when thenucleic acid encoding the desired protein further comprises apromoter/regulatory sequence, the promoter/regulatory is positioned atthe 5′ end of the desired protein coding sequence such that it drivesexpression of the desired protein in a cell. Together, the nucleic acidencoding the desired protein and its promoter/regulatory sequencecomprise a “transgene.” Two polypeptides do not necessarily need to beadjacent to each other in order to be operably linked.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cell under mostor all physiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

A “polyadenylation sequence” is a polynucleotide sequence which directsthe addition of a poly A tail onto a transcribed messenger RNA sequence.A “polynucleotide” means a single strand or parallel and anti-parallelstrands of a nucleic acid. Thus, a polynucleotide may be either asingle-stranded or a double-stranded nucleic acid.

The term “nucleic acid” typically refers to large polynucleotides. Theterm “oligonucleotide” typically refers to short polynucleotides,generally, no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences”; sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

As used herein, the term “therapeutically effective amount” means thequantity of an agent that is effective in treating a condition, disorderor disease.

A “portion” of a polynucleotide means at least at least about twentysequential nucleotide residues of the polynucleotide. It is understoodthat a portion of a polynucleotide may include every nucleotide residueof the polynucleotide. “Primer” refers to a polynucleotide that iscapable of specifically hybridizing to a designated polynucleotidetemplate and providing a point of initiation for synthesis of acomplementary polynucleotide. Such synthesis occurs when thepolynucleotide primer is placed under conditions in which synthesis isinduced, i.e., in the presence of nucleotides, a complementarypolynucleotide template, and an agent for polymerization such as DNApolymerase. A primer is typically single-stranded, but may bedouble-stranded. Primers are typically deoxyribonucleic acids, but awide variety of synthetic and naturally occurring primers are useful formany applications. A primer is complementary to the template to which itis designed to hybridize to serve as a site for the initiation ofsynthesis, but need not reflect the exact sequence of the template. Insuch a case, specific hybridization of the primer to the templatedepends on the stringency of the hybridization conditions. Primers canbe labeled with, e.g., chromogenic, radioactive, or fluorescent moietiesand used as detectable moieties.

“Probe” refers to a polynucleotide that is capable of specificallyhybridizing to a designated sequence of another polynucleotide. A probespecifically hybridizes to a target complementary polynucleotide, butneed not reflect the exact complementary sequence of the template. Insuch a case, specific hybridization of the probe to the target dependson the stringency of the hybridization conditions. Probes can be labeledwith, e.g., chromogenic, radioactive, or fluorescent moieties and usedas detectable moieties.

By “renalase inhibitor” is meant a compound that detectably inhibits thelevel of renalase in a cell or tissue when compared to the level ofrenalase in an otherwise identical cell or tissue in the absence of thecompound. The level of renalase includes, but is not limited to, thelevel of expression of a nucleic acid encoding the molecule, the levelof renalase detectable, and/or the level of renalase activity. Renalaseinhibitors include, but are not limited to, a chemical compound, acofactor, an antibody, a ribozyme, an antisense molecule, a nucleicacid, and the like.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell. A recombinantpolynucleotide may serve a non-coding function (e.g., promoter, originof replication, ribosome-binding site, etc.) as well.

A “recombinant polypeptide” is one which is produced upon expression ofa recombinant polynucleotide.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

The term “protein” typically refers to large polypeptides.

The term “peptide” typically refers to short polypeptides. Conventionalnotation is used herein to portray polypeptide sequences: the left-handend of a polypeptide sequence is the amino-terminus; the right-hand endof a polypeptide sequence is the carboxyl-terminus.

As used herein, the term “renalase” refers to a novel monoamine oxidasesecreted by the kidney that metabolizes biogenic monoamines such asdopamine, norepinephrine, and epinephrine. The renalase moleculesdisclosed herein are a class of molecules that include those having highand/or significant sequence identity with other polypeptides disclosedherein. More specifically, the putative renalase will share at leastabout 40% sequence identity with a nucleic acid having the sequence SEQID NO: 1. More preferably, a nucleic acid encoding renalase has at leastabout 45% identity, or at least about 50% identity, or at least about55% identity, or at least about 60% identity, or at least about 65%identity, or at least about 70% identity, or at least about 75%identity, or at least about 80% identity, or at least about 85%identity, or at least about 90% identity, or at least about 95%identity, or at least about 98%, or at least about 99% sequence identitywith SEQ ID NO: 1 disclosed herein. Even more preferably, the nucleicacid is SEQ ID NO:1.

The term “renalase” also includes renalase isoforms. The renalase genecontains 9 exons spanning 310188 bp in chromosome 10 of human genome.The renalase clone (SEQ ID NO: 1, GenBank accession number: BC005364)disclosed herein is the gene containing exons 1, 2, 3, 4, 5, 6, 8. Thereare at least two additional alternatively-spliced forms of renalaseprotein as shown in the human genome database. One alternatively splicedform contains exons 1, 2, 3, 4, 5, 6, 9, identified by clones in thehuman genome database as GenBank accession number AK002080 (SEQ ID NO:3) and NM_(—)018363 (SEQ ID NO: 4). The other alternatively spliced formcontains exons 5, 6, 7, 8, identified by clones in the human genomedatabase as GenBank accession number BX648154 (SEQ ID NO: 5).

Unless otherwise indicated, “renalase” encompasses all known renalases(e.g., rat renalase, and human renalase), and renalases to bediscovered, including but not limited to, mouse renalase and chimpanzeerenalase, having the characteristics and/or physical features of therenalase disclosed herein.

A “restriction site” is a portion of a double-stranded nucleic acidwhich is recognized by a restriction endonuclease. A portion of adouble-stranded nucleic acid is “recognized” by a restrictionendonuclease if the endonuclease is capable of cleaving both strands ofthe nucleic acid at the portion when the nucleic acid and theendonuclease are contacted.

By the term “specifically binds,” as used herein, is meant a compound,e.g., a protein, a nucleic acid, an antibody, and the like, whichrecognizes and binds a specific molecule, but does not substantiallyrecognize or bind other molecules in a sample.

A first oligonucleotide anneals with a second oligonucleotide “with highstringency” if the two oligonucleotides anneal under conditions wherebyonly oligonucleotides which are at least about 70%, or at least about73%, more preferably, at least about 75%, even more preferably, at leastabout 80%, even more preferably, at least about 85%, yet morepreferably, at least about 90%, and most preferably, at least about 95%,complementary anneal with one another. The stringency of conditions usedto anneal two oligonucleotides is a function of, among other factors,temperature, ionic strength of the annealing medium, the incubationperiod, the length of the oligonucleotides, the G-C content of theoligonucleotides, and the expected degree of non-homology between thetwo oligonucleotides, if known. Methods of adjusting the stringency ofannealing conditions are known (see, e.g., Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York).

As used herein, the term “transgene” means an exogenous nucleic acidsequence which exogenous nucleic acid is encoded by a transgenic cell ormammal.

A “recombinant cell” is a cell that comprises a transgene. Such a cellmay be a eukaryotic cell or a prokaryotic cell. Also, the transgeniccell encompasses, but is not limited to, an embryonic stem cellcomprising the transgene, a cell obtained from a chimeric mammal derivedfrom a transgenic ES cell where the cell comprises the transgene, a cellobtained from a transgenic mammal, or fetal or placental tissue thereof,and a prokaryotic cell comprising the transgene.

By the term “exogenous nucleic acid” is meant that the nucleic acid hasbeen introduced into a cell or an animal using technology which has beendeveloped for the purpose of facilitating the introduction of a nucleicacid into a cell or an animal. By “tag” polypeptide is meant any proteinwhich, when linked by a peptide bond to a protein of interest, may beused to localize the protein, to purify it from a cell extract, toimmobilize it for use in binding assays, or to otherwise study itsbiological properties and/or function.

As used herein, the term “transgenic mammal” means a mammal, the cellsof which comprise an exogenous nucleic acid. The exogenous nucleic acidmay or may not be integrated into the genome of the mammal.

As used herein, to “treat” means reducing the frequency, extent,severity and/or duration with which symptoms of ESRD, hypertension,cardiovascular diseases, mental disorders, and the like, are experiencedby a patient.

By the term “vector” as used herein, is meant any plasmid or virusencoding an exogenous nucleic acid. The term should also be construed toinclude non-plasmid and non-viral compounds which facilitate transfer ofnucleic acid into virions or cells, such as, for example, polylysinecompounds and the like. The vector may be a viral vector which issuitable as a delivery vehicle for delivery of the renalase protein ornucleic acid encoding a mammalian renalase, to the patient, or thevector may be a non-viral vector which is suitable for the same purpose.

Examples of viral and non-viral vectors for delivery of DNA to cells andtissues are well known in the art and are described, for example, in Maet al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94:12744-12746). Examples ofviral vectors include, but are not limited to, a recombinant vacciniavirus, a recombinant adenovirus, a recombinant retrovirus, a recombinantadeno-associated virus, a recombinant avian pox virus, and the like(Cranage et al., 1986, EMBO J. 5:3057-3063; International PatentApplication No. WO94/17810, published Aug. 18, 1994; InternationalPatent Application No. WO94/23744, published Oct. 27, 1994). Examples ofnon-viral vectors include, but are not limited to, liposomes, polyaminederivatives of DNA, and the like.

A “knock-out targeting vector,” as the term is used herein, means avector comprising two nucleic acid sequences each of which iscomplementary to a nucleic acid regions flanking a target sequence ofinterest which is to be deleted and/or replaced by another nucleic acidsequence. The two nucleic acid sequences therefore flank the targetsequence which is to be removed by the process of homologousrecombination.

As used herein, the term “chronic kidney disease” refers to kidneydamage for 3 months as defined by structural or functional abnormalitieswith or without decreased glomerular filtration rate (GFR), or a GFR of60 mL/min/1.73 m² or less, with or without kidney damage. GFR is ameasure of the kidneys' ability to filter blood, which can be expressedon a continuous scale. GFR can be estimated by using the serumcreatinine, the body weight, and age.

As used herein, the term “end stage renal disease (ESRD” refers to acomplete or near complete failure of the kidneys to function to excretewastes, concentrate urine, and regulate electrolytes. End-stage renaldisease (ESRD) occurs when chronic renal failure progresses to the pointat which the kidneys are permanently functioning at less than 10% oftheir capacity. At this point, the kidney function is so low thatwithout dialysis or kidney transplantation, complications are multipleand severe, and death will occur from accumulation of fluids and wasteproducts in the body.

DESCRIPTION

I. Isolated Nucleic Acids

A. Sense Nucleic Acids

The present invention includes an isolated nucleic acid encoding amammalian kidney expressed molecule, renalase, or a fragment thereof,wherein the nucleic acid shares at least about 40% identity with atleast one nucleic acid having the sequence of (SEQ ID NO: 1).Alternatively, the nucleic acid is at least about 45% homologous, or atleast about 50% homologous, or at least about 55% homologous, or atleast about 60% homologous, or at least about 65% homologous, or atleast about 70% homologous, or at least about 75% homologous, or atleast about 80% homologous, or at least about 85% homologous, or atleast about 90% homologous, or at least about 95% homologous, or atleast about 98% homologous, or at least about 99% homologous to SEQ IDNO: 1 disclosed herein. In one embodiment, the nucleic acid sequence ormolecule is provided by the sequence of SEQ ID NO: 1.

In another aspect, the present invention includes an isolated nucleicacid encoding a mammalian renalase, or a fragment thereof, wherein theprotein encoded by the nucleic acid shares greater than about 15%homology with the amino acid sequence of SEQ ID NO: 2. Alignment studiesreveal that renalase has 13.2% amino acid identity with monoamineoxidase A.

Preferably, the protein encoded by the isolated nucleic acid encodingrenalase is at least about 15% homologous, or at least about 20%homologous, or at least about 25% homologous, or at least about 30%homologous, or at least about 35% homologous, or at least about 40%homologous, or at least about 45% homologous, or at least about 50%homologous, or at least about 55% homologous, or at least about 60%homologous, or at least about 65% homologous, or at least about 70%homologous, or at least about 75% homologous, or at least about 80%homologous, or at least about 85% homologous, or at least about 90%homologous, or at least about 95% homologous, or at least about 98%, orat least about 99% homologous to SEQ ID NO: 2. In one embodiment, therenalase polypeptide is encoded by the nucleic acid provide in SEQ IDNO:2. As disclosed herein, a nucleic acid of SEQ ID NO: 1 can betranslated to produce a human renalase protein comprising 342 aminoacids with a calculated molecule mass of 37.8 kDa.

The present invention should not be construed as being limited solely tothe nucleic and amino acid sequences disclosed herein. Once armed withthe present invention, it is readily apparent to one skilled in the artthat other nucleic acids encoding renalase polypeptides such as thosepresent in other species of mammals (e.g., ape, gibbon, bovine, ovine,equine, porcine, canine, feline, murine, and the like), can be obtainedby following the procedures described herein in the experimental detailssection for the isolation of the rat, and human renalase nucleic acidsencoding renalase polypeptides as disclosed herein (e.g., screening ofgenomic or cDNA libraries), and procedures that are well-known in theart (e.g., reverse transcription PCR using mRNA samples) or to bedeveloped.

Further, any number of procedures may be used for the generation ofmutant, derivative or variant forms of renalase using recombinant DNAmethodology well known in the art such as, for example, that describedin Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, New York) and Ausubel et al. (1997,Current Protocols in Molecular Biology, Green & Wiley, New York).

Procedures for the introduction of amino acid changes in a protein orpolypeptide by altering the DNA sequence encoding the polypeptide arewell known in the art and are also described in Sambrook et al. (1989,supra); Ausubel et al. (1997, supra).

The invention further includes a nucleic acid encoding a mammalianrenalase wherein a nucleic acid encoding a tag polypeptide is covalentlylinked thereto. That is, the invention encompasses a chimeric nucleicacid wherein the nucleic acid sequences encoding a tag polypeptide iscovalently linked to the nucleic acid encoding at least one humanrenalase. Such tag polypeptides are well known in the art and include,for instance, green fluorescent protein (GFP), an influenza virushemagglutinin tag polypeptide, myc, myc-pyruvate kinase (myc-PK), His₆,maltose biding protein (MBP), a FLAG tag polypeptide, a HA tagpolypeptide, and a glutathione-S-transferase (GST) tag polypeptide.However, the invention should in no way be construed to be limited tothe nucleic acids encoding the above-listed tag polypeptides. Rather,any nucleic acid sequence encoding a polypeptide which may function in amanner substantially similar to these tag polypeptides should beconstrued to be included in the present invention. The nucleic acidcomprising a nucleic acid encoding a tag polypeptide can be used tolocalize renalase within a cell, a tissue (e.g., a blood vessel, bone,and the like), and/or a whole organism (e.g., an amphibian and/or amammalian embryo, and the like), detect renalase if secreted from acell, and to study the role(s) of renalase in a cell. Further, additionof a tag polypeptide facilitates isolation and purification of the“tagged” protein such that the proteins of the invention can be producedand purified readily.

B. Antisense Nucleic Acids

In certain situations, it may be desirable to inhibit expression ofrenalase and the invention therefore includes compositions useful forinhibition of renalase expression. Thus, the invention features anisolated nucleic acid complementary to a portion or the entire length ofa nucleic acid encoding a mammalian renalase, which nucleic acid is inan antisense orientation with respect to transcription. The antisensenucleic acid is complementary with a nucleic acid having at least about40% homology with SEQ ID NO: 1, or a fragment thereof. In otherembodiments, the antisense nucleic acid is at least about 45%homologous, or at least about 50% homologous, or at least about 55%homologous, or at least about 60% homologous, or at least about 65%homologous, or at least about 70% homologous, or at least about 75%homologous, or at least about 80% homologous, or at least about 85%homologous, or at least about 90% homologous, or at least about 95%homologous, or at least about 99% homologous to a nucleic acidcomplementary to a portion or the entire length of a nucleic acidencoding a mammalian renalase having the sequence of SEQ ID NO: 1, or afragment thereof, which is in an antisense orientation with respect totranscription. Most preferably, the nucleic acid is complementary to aportion or the entire length of a nucleic acid that is SEQ ID NO: 1, ora fragment thereof. Such antisense nucleic acid serves to inhibit theexpression, function, or both, of a kidney expressed (renalase)molecule.

Further, antisense nucleic acids complementary to all or a portion of anucleic acid encoding renalase can be used to detect the expression ofrenalase mRNA in a cell, tissue, and/or organism, using, for example butnot limited to, in situ hybridization. Thus, one skilled in the artwould understand, based upon the disclosure provided herein, that theinvention encompasses antisense nucleic acids that can be used as probesto assess renalase expression.

Antisense molecules of the invention may be made synthetically and thenprovided to the cell. Antisense oligomers of between about 10 to about30, and more preferably about 15 nucleotides, are preferred, since theyare easily synthesized and introduced into a target cell. Syntheticantisense molecules contemplated by the invention includeoligonucleotide derivatives known in the art which have improvedbiological activity compared to unmodified oligonucleotides (see Cohen,supra; Tullis, 1991, U.S. Pat. No. 5,023,243, incorporated by referenceherein in its entirety).

II. Isolated Polypeptides

A. Polypeptides, their Analogs and Modifications

The invention also includes an isolated polypeptide comprising amammalian renalase. In one embodiment, the isolated polypeptidecomprising a mammalian renalase is at least about 15% homologous to apolypeptide having the amino acid sequence of SEQ ID NO:2. Inalternative embodiments, the isolated polypeptide comprising a mammalianrenalase is at least about 20% homologous, or at least about 25%homologous, or at least about 30% homologous, or at least about 35%homologous, or at least about 40% homologous, or at least about 45%homologous, or at least about 50% homologous, or at least about 55%homologous, or at least about 60% homologous, or at least about 65%homologous, or at least about 70% homologous, or at least about 75%homologous, or at least about 80% homologous, or at least about 85%homologous, or at least about 90% homologous, or at least about 95%homologous, or at least about 98% homologous, or at least about 99%homologous to rat renalase. In one embodiment, the isolated polypeptidecomprising a mammalian renalase is that of rat renalase. In anotherembodiment, the isolated polypeptide comprising a mammalian renalasemolecule is SEQ ID NO:2.

The present invention also provides for analogs of proteins or peptideswhich comprise a renalase as disclosed herein. Analogs may differ fromnaturally occurring proteins or peptides by conservative amino acidsequence differences or by modifications which do not affect sequence,or by both. For example, conservative amino acid changes may be made,which although they alter the primary sequence of the protein orpeptide, do not normally alter its function. Conservative amino acidsubstitutions typically include substitutions within the followinggroups:

-   -   glycine, alanine;    -   valine, isoleucine, leucine;    -   aspartic acid, glutamic acid;    -   asparagine, glutamine;    -   serine, threonine;    -   lysine, arginine;    -   phenylalanine, tyrosine.

Modifications (which do not normally alter primary sequence) include invivo, or in vitro, chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps; e.g., by exposing the polypeptide to enzymes whichaffect glycosylation, e.g., mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences which have phosphorylated aminoacid residues, e.g., phosphotyrosine, phosphoserine, orphosphothreonine.

Also included are polypeptides which have been modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. Analogs of suchpolypeptides include those containing residues other than naturallyoccurring L-amino acids, e.g., D-amino acids or non-naturally occurringsynthetic amino acids. The peptides of the invention are not limited toproducts of any of the specific exemplary processes listed herein.

The present invention should also be construed to encompass “mutants,”“derivatives,” and “variants” of the peptides of the invention (or ofthe DNA encoding the same) which mutants, derivatives and variants arerenalase peptides which are altered in one or more amino acids (or, whenreferring to the nucleotide sequence encoding the same, are altered inone or more base pairs) such that the resulting peptide (or DNA) is notidentical to the sequences recited herein, but has the same biologicalproperty as the peptides disclosed herein, in that the peptide hasbiological/biochemical properties of the renalase peptide of the presentinvention.

Further, the invention should be construed to include renalase isoforms,and naturally occurring variants or recombinantly derived mutants ofrenalase sequences, which variants or mutants render the protein encodedthereby either more, less, or just as biologically active as thefull-length clones of the invention.

B. Production and Isolation of Polypeptides from Cells

As demonstrated elsewhere herein, the present invention also relates tomethods for the production and isolation of renalase polypeptides fromcells that produce renalase. The invention also contemplates methods forthe production and isolation of renalase from the cellular media inwhich such cells are grown.

Cells that can be used in such methods include any cells that naturallyproduce renalase and cells that have been mutated, altered, or treatedso as to produce renalase. Such cells include those that produce amountsof renalase typical for that type of cell and those cells thatoverproduce renalase. Cells that do not naturally produce renalase orproduce renalase in low amounts may be mutated, altered or treated sothat they produce renalase in amounts physically and/or economicallypractical for its isolation from such cells and the media in which theyare grown.

Once produced by the cells, the renalase can be isolated from the cellsand/or their growth media using protein isolation techniques well knownto those skilled in the art of protein isolation. If desired ornecessary, the isolated renalase may be further purified usingpurification techniques well known to those skilled in the art ofprotein purification.

C. Purification of Polypeptides from Bodily Fluids

The present invention also relates to a method for the purification ofrenalase polypeptides from bodily fluids of animals, particularlymammals. Any animal that produces renalase can be used for thepurification of renalase from its bodily fluids. Examples of suitableanimals include but are not limited to mice, rats, horses, pigs, dogs,monkeys, cows, and humans.

Purification of the polypeptides including fragments, homologouspolypeptides, muteins, analogs, derivatives and fusion proteins iswell-known and within the skill of one having ordinary skill in the art.See, e.g., Scopes, Protein Purification, 2d ed. (1987). Purification ofchemically-synthesized peptides can readily be effected, e.g., by HPLC.Accordingly, the present invention provides a method of purifying arenalase polypeptide from at least one bodily fluid. The bodily fluidsinclude, but are not limited to, blood, serum, plasma, saliva, urine,lymph fluid, whole blood, spinal fluid tissue culture medium, andcellular extracts. Purification of renalase from bodily fluids may beconducted by any protein purification known in the art, including butare not limited to, procedures of ion exchange chromatography,adsorption chromatography, ligand-bound affinity chromatography and gelpermeation chromatography, solely or in combination.

It is an aspect of the present invention to provide the isolatedproteins of the present invention in pure or substantially pure form inthe presence or absence of a stabilizing agent. Stabilizing agentsinclude both proteinaceous or non-proteinaceous material and arewell-known in the art. Stabilizing agents, such as albumin andpolyethylene glycol (PEG) are known and are commercially available.

Although high levels of purity are preferred when the isolated proteinsof the present invention are used as therapeutic agents, such as invaccines and as replacement therapy, the isolated proteins of thepresent invention are also useful at lower purity. For example,partially purified proteins of the present invention can be used asimmunogens to raise antibodies in laboratory animals.

D. Activity of Polypeptides

The present invention further provides a pharmaceutical compositioncomprising a cofactor for enzyme activation. As more fully disclosedelsewhere herein, renalase is a flavin-adenosine-dinucleotide(FAD)-containing enzyme that requires the cofactor FAD for itsfunctionality. Once armed with the present invention, it is readilyapparent to one skilled in the art that other enzyme cofactors such asthose which may function in a manner substantially similar to FAD andthose well-known in the art can be employed to activate or deactivaterenalase activity. These cofactors include, but are not limited to, FADanalogs, nicotinamide adenine dinucleotide (NAD), nicotinamide adeninedinucleotide phosphate (NADP), thiamine pyrophosphate, flavin adeninedinucleotide (FAD), flavin mononucleotide (FMN), pyridoxal phosphate,coenzyme A, tetrahydrofolate, adenosine triphosphate, guanosinetriphosphate and S-adenosyl methionine (SAM), metal ion, metalporphyrin, e.g. heme groups, biotin, α2-microglobulin, thiaminepyrophosphate, coenzyme A, pyridoxal phosphate, coenzyme B12, biocytine,tetrahydrofolate, and lipoic acid.

Renalase activity may also be regulated by the formation of ahomomultimer or a heteromultimer. A homomultimer may be a polypeptideconsisting of three or more identical subunits. On the other hand, themultimeric polypeptide may be a heterodimer, i.e. a polypeptideconsisting of two different subunits, or a heteromultimer consisting ofthree or more subunits wherein at least two of these subunits aredifferent. For example, the multimeric polypeptide is comprised of aplurality of subunits which form a “single” multimeric polypeptide or acomplex of a plurality of functionally associated polypeptides which mayin turn be monomeric and/or multimeric polypeptides.

Homodimers or homomultimers may be formed by a spontaneous associationof several identical polypeptide subunits. Heterodimers orheteromultimers may be formed by a spontaneous association of severaldifferent polypeptide subunits. There is evidence that renalase forms adimer or multidimer complex (FIG. 6C). It is believed that dimerizationor multimerization of renalase may positively or negatively affect theenzyme activity. Accordingly, it is expected that disruption orstabilization of the dimer or multimer complex of renalase may beparticularly useful for various therapeutic purposes.

E. Uses of Polypeptides

The nucleic acids, and peptides encoded thereby, are useful tools forelucidating the function(s) of renalase molecules in a cell. Further,nucleic and amino acids comprising mammalian renalase molecules areuseful targets that can be used, for example, to identify a compoundthat affects renalase expression, and the like, and is a potentialtherapeutic drug candidate for high blood pressure, kidney diseases,heart diseases and the like. The nucleic acids, the proteins encodedthereby, or both, can be administered to a mammal to increase ordecrease expression or activity of renalase in the mammal. This can bebeneficial for the mammal in situations where under or over-expressionof renalase in the mammal mediates a disease or condition associatedwith altered expression of renalase compared with normal expression ofrenalase in a healthy mammal. Such conditions that can be affected bymodulating renalase expression or activity thereby providing atherapeutic benefit include, but are not limited to, high bloodpressure, kidney diseases, heart diseases and the like. This is because,as more fully disclosed elsewhere herein, infusion of renalase in ratshas been shown to lower the blood pressure and heart rate.

Additionally, the nucleic and amino acids of the invention can be usedto produce recombinant cells and transgenic non-human mammals which areuseful tools for the study of renalase action, the identification ofnovel diagnostics and therapeutics for treatment, and for elucidatingthe cellular role(s) of renalase, among other things. For instance,transgenic animals can be used to study kidney and vascular diseaserelated conditions.

Further, the nucleic and amino acids of the invention can be useddiagnostically, either by assessing the level of gene expression or thelevel of protein expression, to assess severity and prognosis of ESRD,high blood pressure, heart diseases, kidney diseases, cardiovasculardiseases, and the like. The nucleic acids, peptides, polypeptides andproteins of the invention are also useful in the development of assaysto assess the efficacy of a treatment for preventing ESRD, high bloodpressure, cardiovascular diseases, and the like. That is, the nucleicacids, peptides, polypeptides and proteins of the invention can be usedto detect the effect of various therapies on renalase expression,thereby ascertaining the effectiveness of the therapies such as, but notlimited to, assessment of treatment efficacies for ESRD, high bloodpressure, heart diseases, kidney diseases, and cardiovascular diseases.

F. Small Molecule Inhibitors of the Polypeptides

In addition to antibodies, ribozymes, interfering RNA's (i.e., RNAi),and antisense nucleic acid molecules as disclosed herein, the presentinvention further provides methods of using small molecules to modulaterenalase activity. As used herein, the term “potential small moleculeinhibitor” refers to a small molecule which binds to a selected proteinbut for which the ability to inhibit a biological activity (e.g., reducethe catalytic rate of an enzyme) of the enzyme has not yet been tested.Following confirmation of such inhibitory characteristic, the smallmolecule can be referred to as a “small molecule inhibitor” or, moregenerally, an “inhibitor”.

The term “small molecule” refers to a compound which has a molecularmass equal to or less than about 5000 Daltons (5 kD), or less than about3 kD, or less than about 2 kD, or less than about 1 kD. In some cases itis preferred that a small molecule have a molecular mass equal to orless than about 700 Da.

As provided in the Examples, the proteins and nucleic acids of theinvention, such as the protein having the amino acid sequence of SEQ IDNO. 2, are involved in catecholamine metabolism. Small molecules thatmodulate or down-regulate the expression of the protein or agents suchas agonists or antagonists of at least one activity of the protein maybe used to modulate biological and pathologic processes associated withthe protein's function and activity.

Pathological processes refer to a category of biological processes whichproduce a deleterious effect. For example, lack of expression ordown-regulation of expression of a protein of the invention may beassociated with certain diseases such as ESRD. As used herein, a smallmolecule inhibitor is said to modulate a pathological process when theagent reduces the degree or severity of the process. For instance, adisease may be prevented or disease progression modulated by theadministration of agents which reduce or modulate in some way theexpression or at least one activity of a protein of the invention.

The small molecule inhibitor of the present invention can be providedalone, or in combination with other agents that modulate a particularpathological process. As used herein, two small molecules are said to beadministered in combination when the two small molecules areadministered simultaneously or are administered independently in afashion such that the agents will act at the same time.

The small molecule inhibitors of the present invention can beadministered via parenteral, subcutaneous, intravenous, intramuscular,intraperitoneal, transdermal, or buccal routes. Alternatively, orconcurrently, administration may be by the oral route. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired.

III. Vectors

A. Vectors for In Vitro Expression

In other related aspects, the invention includes an isolated nucleicacid encoding a mammalian renalase operably linked to a nucleic acidcomprising a promoter/regulatory sequence such that the nucleic acid ispreferably capable of directing expression of the protein encoded by thenucleic acid. Thus, the invention encompasses expression vectors andmethods for the introduction of exogenous DNA into cells withconcomitant expression of the exogenous DNA in the cells such as thosedescribed, for example, in Sambrook et al. (1989, supra), and Ausubel etal. (1997, supra).

Expression of renalase, either alone or fused to a detectable tagpolypeptide, in cells which either do not normally express the renalaseor which do not express renalase fused with a tag polypeptide, may beaccomplished by generating a plasmid, viral, or other type of vectorcomprising the desired nucleic acid operably linked to apromoter/regulatory sequence which serves to drive expression of theprotein, with or without tag, in cells in which the vector isintroduced. Many promoter/regulatory sequences useful for drivingconstitutive expression of a gene are available in the art and include,but are not limited to, for example, the cytomegalovirus immediate earlypromoter enhancer sequence, the SV40 early promoter, both of which wereused in the experiments disclosed herein, as well as the Rous sarcomavirus promoter, and the like. Moreover, inducible and tissue specificexpression of the nucleic acid encoding renalase may be accomplished byplacing the nucleic acid encoding renalase, with or without a tag, underthe control of an inducible or tissue specific promoter/regulatorysequence. Examples of tissue specific or inducible promoter/regulatorysequences which are useful for his purpose include, but are not limitedto the MMTV LTR inducible promoter, and the SV40 late enhancer/promoter.In addition, promoters which are well known in the art which are inducedin response to inducing agents such as metals, glucocorticoids, and thelike, are also contemplated in the invention. Thus, it will beappreciated that the invention includes the use of anypromoter/regulatory sequence, which is either known or unknown, andwhich is capable of driving expression of the desired protein operablylinked thereto.

Expressing renalase using a vector allows the isolation of large amountsof recombinantly produced protein. Further, where the lack or decreasedlevel of renalase expression causes a disease, disorder, or conditionassociated with such expression, the expression of renalase driven by apromoter/regulatory sequence can provide useful therapeutics including,but not limited to, gene therapy whereby renalase is provided. Adisease, disorder or condition associated with a increased level ofexpression, level of protein, or decreased activity of the protein, forwhich administration of renalase can be useful therapeutics including,but not limited to, gene therapy whereby renalase is provided.Therefore, the invention includes not only methods of inhibitingrenalase expression, translation, and/or activity, but it also includesmethods relating to increasing renalase expression, protein level,and/or activity since both decreasing and increasing renalase expressionand/or activity can be useful in providing effective therapeutics.

Selection of any particular plasmid vector or other DNA vector is not alimiting factor in this invention and a wide variety of vectors arewell-known in the art. Further, it is well within the skill of theartisan to choose particular promoter/regulatory sequences and operablylink those promoter/regulatory sequences to a DNA sequence encoding adesired polypeptide. Such technology is well known in the art and isdescribed, for example, in Sambrook, supra, and Ausubel, supra.

The invention thus includes a vector comprising an isolated nucleic acidencoding a mammalian renalase. The incorporation of a desired nucleicacid into a vector and the choice of vectors is well-known in the art asdescribed in, for example, Sambrook et al., supra, and Ausubel et al.,supra.

The invention also includes cells, viruses, proviruses, and the like,containing such vectors. Methods for producing cells comprising vectorsand/or exogenous nucleic acids are well-known in the art. See, e.g.,Sambrook et al., supra; Ausubel et al., supra.

The nucleic acids encoding renalase can be cloned into various plasmidvectors. However, the present invention should not be construed to belimited to plasmids or to any particular vector. Instead, the presentinvention should be construed to encompass a wide plethora of vectorswhich are readily available and/or well-known in the art and no vectorat all.

B. Vectors for In Vivo and Ex Vivo Expression

The renalase polypeptides may also be employed in accordance with thepresent invention by expression of such polypeptides in vivo, which isoften referred to as “gene therapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art and are apparentfrom the teachings herein. For example, cells may be engineered by theuse of a retroviral plasmid vector containing RNA encoding a polypeptideof the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Forexample, a packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

The present invention contemplates the use of any of a variety ofvectors for introduction of constructs comprising the coding sequencefor two or more polypeptides or proteins and a self processing cleavagesequence into cells such that protein expression results. Numerousexamples of expression vectors are known in the art and may be of viralor non-viral origin. Non-viral gene delivery methods which may beemployed in the practice of the invention, include but are not limitedto plasmids, liposomes, nucleic acid/liposome complexes, cationic lipidsand the like.

Viral vectors can efficiently transduce cells and introduce their ownDNA into a host cell. In generating recombinant viral vectors,non-essential genes are replaced with a gene encoding a protein orpolypeptide of interest. Exemplary vectors include but are not limitedto viral and non-viral vectors, such a retroviral vector (includinglentiviral vectors), adenoviral (Ad) vectors including replicationcompetent, replication deficient and gutless forms thereof,adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors,bovine papilloma vectors, Epstein-Barr vectors, herpes vectors, vacciniavectors, Moloney murine leukemia vectors, Harvey murine sarcoma virusvectors, murine mammary tumor virus vectors, Rous sarcoma virus vectorsand nonviral plasmids.

The vector typically comprises an origin of replication and the vectormay or may not in addition comprise a “marker” or “selectable marker”function by which the vector can be identified and selected. While anyselectable marker can be used, selectable markers for use in recombinantvectors are generally known in the art and the choice of the properselectable marker will depend on the host cell. Examples of selectablemarker genes which encode proteins that confer resistance to antibioticsor other toxins include, but are not limited to ampicillin,methotrexate, tetracycline, neomycin (Southern et al., J., J Mol ApplGenet. 1982; 1(4):327-41 (1982)), mycophenolic acid (Mulligan et al.,Science 209:1422-7 (1980)), puromycin, zeomycin, hygromycin (Sugden etal., Mol Cell Biol. 5(2):410-3 (1985)) and G418. As will be understoodby those of skill in the art, expression vectors typically include anorigin of replication, a promoter operably linked to the coding sequenceor sequences to be expressed, as well as ribosome binding sites, RNAsplice sites, a polyadenylation site, and transcriptional terminatorsequences, as appropriate to the coding sequence(s) being expressed.

Reference to a vector or other DNA sequences as “recombinant” merelyacknowledges the operable linkage of DNA sequences that are nottypically operably linked as isolated from or found in nature.Regulatory (expression and/or control) sequences are operatively linkedto a nucleic acid coding sequence when the expression and/or controlsequences regulate the transcription and, as appropriate, translation ofthe nucleic acid sequence. Thus expression and/or control sequences caninclude promoters, enhancers, transcription terminators, a start codon(i.e., ATG) 5′ to the coding sequence, splicing signals for introns andstop codons.

Adenovirus gene therapy vectors are known to exhibit strong transientexpression, excellent titer, and the ability to transduce dividing andnon-dividing cells in vivo (Hitt et al., Adv in Virus Res 55:479-505,2000). The recombinant Ad vectors of the instant invention comprise: (1)a packaging site enabling the vector to be incorporated intoreplication-defective Ad virions; (2) the coding sequence for two ormore proteins or polypeptide of interest, and (3) a sequence encoding aself-processing cleavage site alone or in combination with an additionalproteolytic cleavage site. Other elements necessary or helpful forincorporation into infectious virions, include the 5′ and 3′ Ad ITRs,the E2 genes, portions of the E4 gene and optionally the E3 gene.

Replication-defective Ad virions encapsulating the recombinant Advectors of the instant invention are made by standard techniques knownin the art using Ad packaging cells and packaging technology. Examplesof these methods may be found, for example, in U.S. Pat. No. 5,872,005.The coding sequence for two or more polypeptides or proteins of interestis commonly inserted into adenovirus in the deleted E3 region of thevirus genome. Preferred adenoviral vectors for use in practicing theinvention do not express one or more wild-type Ad gene products, e.g.,E1a, E1b, E2, E3, and E4. Preferred embodiments are virions that aretypically used together with packaging cell lines that complement thefunctions of E1, E2A, E4 and optionally the E3 gene regions. See, e.g.U.S. Pat. Nos. 5,872,005, 5,994,106, 6,133,028 and 6,127,175. Thus, asused herein, “adenovirus” and “adenovirus particle” refer to the virusitself or derivatives thereof and cover all serotypes and subtypes andboth naturally occurring and recombinant forms, except where indicatedotherwise. Such adenoviruses may be wildtype or may be modified invarious ways known in the art or as disclosed herein. Such modificationsinclude modifications to the adenovirus genome that is packaged in theparticle in order to make an infectious virus. Such modificationsinclude deletions known in the art, such as deletions in one or more ofthe E1a, E1b, E2a, E2b, E3, or E4 coding regions. Exemplary packagingand producer cells are derived from 293, A549 or HeLa cells. Adenovirusvectors are purified and formulated using standard techniques known inthe art.

Adeno-associated virus (AAV) is a helper-dependent human parvovirus thatis able to infect cells latently by chromosomal integration. Because ofits ability to integrate chromosomally and its nonpathogenic nature, AAVhas significant potential as a human gene therapy vector. For use inpracticing the present invention rAAV virions are produced usingstandard methodology, known to those of skill in the art and areconstructed such that they include, as operatively linked components inthe direction of transcription, control sequences includingtranscriptional initiation and termination sequences, and the codingsequence(s) of interest. More specifically, the recombinant AAV vectorsof the instant invention comprise: (1) a packaging site enabling thevector to be incorporated into replication-defective AAV virions; (2)the coding sequence for two or more proteins or polypeptide of interest;(3) a sequence encoding a self-processing cleavage site alone or incombination with an additional proteolytic cleavage site. AAV vectorsfor use in practicing the invention are constructed such that they alsoinclude, as operatively linked components in the direction oftranscription, control sequences including transcriptional initiationand termination sequences. These components are flanked on the 5′ and 3′end by functional AAV ITR sequences. By “functional AAV ITR sequences”is meant that the ITR sequences function as intended for the rescue,replication and packaging of the AAV virion.

Recombinant AAV vectors are also characterized in that they are capableof directing the expression and production of selected recombinantproteins or polypeptides of interest in target cells. Thus, therecombinant vectors comprise at least all of the sequences of AAVessential for encapsidation and the physical structures for infection ofthe recombinant AAV (rAAV) virions. Hence, AAV ITRs for use in thevectors of the invention need not have a wild-type nucleotide sequence(e.g., as described in Kotin, Hum. Gene Ther., 5:793-801, 1994), and maybe altered by the insertion, deletion or substitution of nucleotides orthe AAV ITRs may be derived from any of several AAV serotypes.Generally, an AAV vector is a vector derived from an adeno-associatedvirus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV4,AAV-5, AAV-6, AAV-7, AAV-8, etc. Preferred rAAV expression vectors havethe wild type REP and CAP genes deleted in whole or part, but retainfunctional flanking ITR sequences.

Typically, an AAV expression vector is introduced into a producer cell,followed by introduction of an AAV helper construct, where the helperconstruct includes AAV coding regions capable of being expressed in theproducer cell and which complement AAV helper functions absent in theAAV expression vector. As used herein, the term “AAV helper functions”refers to AAV coding regions capable of being expressed in the host cellto complement AAV viral functions missing from the rAAV vector.Typically, the AAV helper functions include the AAV rep coding regionand the AAV cap coding region. The helper construct may be designed todown regulate the expression of the large Rep proteins (Rep78 andRep68), typically by mutating the start codon following p5 from ATG toACG, as described in U.S. Pat. No. 6,548,286.

Introduction of an AAV expression vector into a producer cell istypically followed by introduction of helper virus and/or additionalvectors into the producer cell, wherein the helper virus and/oradditional vectors provide accessory functions capable of supportingefficient rAAV virus production.

“Accessory functions” refer to functions that are required by AAV forreplication, but are not provided by the AAV virion itself. Thus, theseaccessory functions and factors must be provided by the host cell, avirus (e.g., adenovirus, herpes simplex virus or vaccinia virus), or byan expression vector that is co-expressed in the same cell. Generally,the E1A and E1B, E2A, E4 and VA coding regions of adenovirus are used tosupply the necessary accessory function required for AAV replication andpackaging (Matsushita et al., Gene Therapy 5:938 [1998]).

The producer cells are then cultured to produce rAAV. These steps arecarried out using standard methodology. Replication-defective AAVvirions encapsulating the recombinant AAV vectors of the instantinvention are made by standard techniques known in the art using AAVpackaging cells and packaging technology. Examples of these methods maybe found, for example, in U.S. Pat. Nos. 5,436,146; 5,753,500,6,040,183, 6,093,570 and 6,548,286. Further compositions and methods forpackaging are described in Wang et al. (US 2002/0168342) and includethose techniques within the knowledge of those of skill in the art. BothAAV vectors and AAV helper constructs can be constructed to contain oneor more optional selectable marker genes. Selectable marker genes whichconfer antibiotic resistance or sensitivity to an appropriate selectivemedium are generally known in the art.

The term “AAV virion” refers to a complete virus particle, such as a“wild-type” (wt) AAV virus particle (comprising a linear,single-stranded AAV nucleic acid genome associated with an AAV capsidprotein coat). In contrast, a “recombinant AAV virion,” and “rAAVvirion” refers to an infectious viral particle containing a heterologousDNA sequence of interest, flanked on both sides by AAV ITRs.

In practicing the invention, host cells for producing rAAV virionsinclude mammalian cells, insect cells, microorganisms and yeast. Hostcells can also be packaging cells in which the AAV rep and cap genes arestably maintained in the host cell or producer cells in which the AAVvector genome is stably maintained and packaged. Exemplary packaging andproducer cells are derived from 293, A549 or HeLa cells. AAV vectors arepurified and formulated using standard techniques known in the art.

Retroviral vectors are also a common tool for gene delivery (Miller,Nature 357: 455-460, 1992). Retroviral vectors and more particularlylentiviral vectors may be used in practicing the present invention.Accordingly, the term “retrovirus” or “retroviral vector”, as usedherein is meant to include “lentivirus” and “lentiviral vectors”respectively. Retroviral vectors have been tested and found to besuitable delivery vehicles for the stable introduction of genes ofinterest into the genome of a broad range of target cells. The abilityof retroviral vectors to deliver unrearranged, single copy transgenesinto cells makes retroviral vectors well suited for transferring genesinto cells. Further, retroviruses enter host cells by the binding ofretroviral envelope glycoproteins to specific cell surface receptors onthe host cells. Consequently, pseudotyped retroviral vectors in whichthe encoded native envelope protein is replaced by a heterologousenvelope protein that has a different cellular specificity than thenative envelope protein (e.g., binds to a different cell-surfacereceptor as compared to the native envelope protein) may also findutility in practicing the present invention. The ability to direct thedelivery of retroviral vectors encoding one or more target proteincoding sequences to specific target cells is desirable in practice ofthe present invention.

The present invention provides retroviral vectors which include e.g.,retroviral transfer vectors comprising one or more transgene sequencesand retroviral packaging vectors comprising one or more packagingelements. In particular, the present invention provides pseudotypedretroviral vectors encoding a heterologous or functionally modifiedenvelope protein for producing pseudotyped retrovirus.

The core sequence of the retroviral vectors of the present invention maybe readily derived from a wide variety of retroviruses, including forexample, B, C, and D type retroviruses as well as spumaviruses andlentiviruses (RNA Tumor Viruses, Second Edition, Cold Spring HarborLaboratory, 1985). An example of a retrovirus suitable for use in thecompositions and methods of the present invention includes, but is notlimited to, a lentivirus. Other retroviruses suitable for use in thecompositions and methods of the present invention include, but are notlimited to, Avian Leukosis Virus, Bovine Leukemia Virus, Murine LeukemiaVirus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus and Rous Sarcoma Virus. Preferred MurineLeukemia Viruses include 4070A and 1504A (Hartley and Rowe, J. Virol.19:19-25, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245),Graffi, Gross (ATCC No. VR-590), Kirsten, Harvey Sarcoma Virus andRauscher (ATCC No. VR-998), and Moloney Murine Leukemia Virus (ATCC No.VR-190). Such retroviruses may be readily obtained from depositories orcollections such as the American Type Culture Collection (“ATCC”;Rockville, Md.), or isolated from known sources using commonly availabletechniques.

Preferably, a retroviral vector sequence of the present invention isderived from a lentivirus. A preferred lentivirus is a humanimmunodeficiency virus, e.g., type 1 or 2 (i.e., HIV-1 or HIV-2, whereinHIV-1 was formerly called lymphadenopathy associated virus 3 (HTLV-III)and acquired immune deficiency syndrome (AIDS)-related virus (ARV)), oranother virus related to HIV-1 or HIV-2 that has been identified andassociated with AIDS or AIDS-like disease. Other lentiviruses include asheep Visna/maedi virus, a feline immunodeficiency virus (FIV), a bovinelentivirus, simian immunodeficiency virus (SIV), an equine infectiousanemia virus (EIAV), and a caprine arthritis-encephalitis virus (CAEV).

The various genera and strains of retroviruses suitable for use in thecompositions and methods are well known in the art (see, e.g., FieldsVirology, Third Edition, edited by B. N. Fields et al., Lippincott-RavenPublishers (1996), see e.g., Chapter 58, Retroviridae: The Viruses andTheir Replication, Classification, pages 1768-1771.

The packaging systems of the present invention comprise at least twopackaging vectors, a first packaging vector which comprises a firstnucleotide sequence comprising a gag, a pol, or gag and pol genes and asecond packaging vector which comprises a second nucleotide sequencecomprising a heterologous or functionally modified envelope gene. In apreferred embodiment, the retroviral elements are derived from alentivirus, such as HIV. Preferably, the vectors lack a functional tatgene and/or functional accessory genes (vif, vpr, vpu, vpx, nef). In afurther preferred embodiment, the system further comprises a thirdpackaging vector that comprises a nucleotide sequence comprising a revgene. The packaging system can be provided in the form of a packagingcell that contains the first, second, and, optionally, third nucleotidesequences.

The invention is applicable to a variety of systems, and those skilledin the art will appreciate the common elements shared across differinggroups of retroviruses. The description herein uses lentiviral systemsas a representative example. However, all retroviruses share thefeatures of enveloped virions with surface projections and containingone molecule of linear, positive-sense single stranded RNA, a genomeconsisting of a dimer, and the common proteins gag, pol and env.

Lentiviruses share several structural virion proteins in common,including the envelope glycoproteins SU (gp120) and TM (gp41), which areencoded by the env gene; CA (p24), MA (p17) and NC (p7-11), which areencoded by the gag gene; and RT, PR and IN encoded by the pol gene.HIV-1 and HIV-2 contain accessory and other proteins involved inregulation of synthesis and processing virus RNA and other replicativefunctions. The accessory proteins, encoded by the vif, vpr, vpu/vpx, andnef genes, can be omitted (or inactivated) from the recombinant system.In addition, tat and rev can be omitted or inactivated, e.g., bymutation or deletion.

First generation lentiviral vector packaging systems provide separatepackaging constructs for gag/pol and env, and typically employ aheterologous or functionally modified envelope protein for safetyreasons. In second generation lentiviral vector systems, the accessorygenes, vif, vpr, vpu and nef, are deleted or inactivated. Thirdgeneration lentiviral vector systems are preferred for use in practicingthe present invention and include those from which the tat gene has beendeleted or otherwise inactivated (e.g., via mutation).

Compensation for the regulation of transcription normally provided bytat can be provided by the use of a strong constitutive promoter, suchas the human cytomegalovirus immediate early (HCMV-IE)enhancer/promoter. Other promoters/enhancers can be selected based onstrength of constitutive promoter activity, specificity for targettissue (e.g., a liver-specific promoter), or other factors relating todesired control over expression, as is understood in the art. Forexample, in some embodiments, it is desirable to employ an induciblepromoter such as tet to achieve controlled expression. The gene encodingrev is preferably provided on a separate expression construct, such thata typical third generation lentiviral vector system will involve fourplasmids: one each for gagpol, rev, envelope and the transfer vector.Regardless of the generation of packaging system employed, gag and polcan be provided on a single construct or on separate constructs.

Typically, the packaging vectors are included in a packaging cell, andare introduced into the cell via transfection, transduction orinfection. Methods for transfection, transduction or infection are wellknown by those of skill in the art. A retroviral/lentiviral transfervector of the present invention can be introduced into a packaging cellline, via transfection, transduction or infection, to generate aproducer cell or cell line.

The packaging vectors of the present invention can be introduced intohuman cells or cell lines by standard methods including, e.g., calciumphosphate transfection, lipofection or electroporation. In someembodiments, the packaging vectors are introduced into the cellstogether with a dominant selectable marker, such as neo, DHFR, Ginsynthetase or ADA, followed by selection in the presence of theappropriate drug and isolation of clones. A selectable marker gene canbe linked physically to genes encoding by the packaging vector.

Stable cell lines, wherein the packaging functions are configured to beexpressed by a suitable packaging cell, are known. For example, see U.S.Pat. No. 5,686,279; and Ory et al., Proc. Natl. Acad. Sci. (1996)93:11400-11406, which describe packaging cells. Further description ofstable cell line production can be found in Dull et al., 1998, J.Virology 72(11):8463-8471; and in Zufferey et al., 1998, J. Virology72(12):9873-9880.

Zufferey et al., 1997, Nature Biotechnology 15:871-875, teach alentiviral packaging plasmid wherein sequences 3′ of pol including theHIV-1 envelope gene are deleted. The construct contains tat and revsequences and the 3′ LTR is replaced with poly A sequences. The 5′ LTRand psi sequences are replaced by another promoter, such as one which isinducible. For example, a CMV promoter or derivative thereof can beused.

Preferred packaging vectors may contain additional changes to thepackaging functions to enhance lentiviral protein expression and toenhance safety. For example, all of the HIV sequences upstream of gagcan be removed. Also, sequences downstream of the envelope can beremoved. Moreover, steps can be taken to modify the vector to enhancethe splicing and translation of the RNA.

Optionally, a conditional packaging system is used, such as thatdescribed by Dull et al., J. Virology 72(11):8463-8471, 1998. Alsopreferred is the use of a self-inactivating vector (SIN), which improvesthe biosafety of the vector by deletion of the HIV-1 long terminalrepeat (LTR) as described, for example, by Zufferey et al., 1998, J.Virology 72(12):9873-9880. Inducible vectors can also be used, such asthrough a tet-inducible LTR.

Herpes simplex virus (HSV) has generated considerable interest intreating nervous system disorders due to its tropism for neuronal cells,but this vector also can be exploited for other tissues given its widehost range. Another factor that makes HSV an attractive vector is thesize and organization of the genome. Because HSV is large, incorporationof multiple genes or expression cassettes is less problematic than inother smaller viral systems. In addition, the availability of differentviral control sequences with varying performance (temporal, strength,etc.) makes it possible to control expression to a greater extent thanin other systems. It also is an advantage that the virus has relativelyfew spliced messages, further easing genetic manipulations.

HSV also is relatively easy to manipulate and can be grown to hightiters. Thus, delivery is less of a problem, both in terms of volumesneeded to attain sufficient MOI and in a lessened need for repeatdosings. For a review of HSV as a gene therapy vector, see Glorioso etal. (1995). A person of ordinary skill in the art would be familiar withwell-known techniques for use of HSV as vectors.

Vaccinia virus vectors have been used extensively because of the ease oftheir construction, relatively high levels of expression obtained, widehost range and large capacity for carrying DNA. Vaccinia contains alinear, double-stranded DNA genome of about 186 kb that exhibits amarked “A-T” preference. Inverted terminal repeats of about 10.5 kbflank the genome. The majority of essential genes appear to map withinthe central region, which is most highly conserved among poxviruses.Estimated open reading frames in vaccinia virus number from 150 to 200.Although both strands are coding, extensive overlap of reading frames isnot common.

Other viral vectors may be employed as constructs in the presentinvention. For example, vectors derived from viruses such as poxvirusmay be employed. A molecularly cloned strain of Venezuelan equineencephalitis (VEE) virus has been genetically refined as a replicationcompetent vaccine vector for the expression of heterologous viralproteins (Davis et al., 1996). Studies have demonstrated that VEEinfection stimulates potent CTL responses and has been suggested thatVEE may be an extremely useful vector for immunizations (Caley et al.,1997). It is contemplated in the present invention, that VEE virus maybe useful in targeting dendritic cells.

A polynucleotide may be housed within a viral vector that has beenengineered to express a specific binding ligand. The virus particle willthus bind specifically to the cognate receptors of the target cell anddeliver the contents to the cell. A novel approach designed to allowspecific targeting of retrovirus vectors was developed based on thechemical modification of a retrovirus by the chemical addition oflactose residues to the viral envelope. This modification can permit thespecific infection of hepatocytes via sialoglycoprotein receptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et al., 1989).

Any vector for use in practicing the invention will include heterologouscontrol sequences, such as a constitutive promoter, e.g., thecytomegalovirus (CMV) immediate early promoter, the RSV LTR, the MoMLVLTR, and the PGK promoter; tissue or cell type specific promotersincluding mTTR, TK, HBV, HAAT, regulatable or inducible promoters,enhancers, etc. Preferred promoters include the LSP promoter (III etal., Blood Coagul. Fibrinolysis 8S2:23-30, 1997), the EF1-alpha promoter(Kim et al., Gene 91(2):217-23, 1990) and Guo et al., Gene Ther.3(9):802-10, 1996). Most preferred promoters include the elongationfactor 1-alpha (EF1a) promoter, a phosphoglycerate kinase-1 (PGK)promoter, a cytomegalovirus immediate early gene (CMV) promoter,chimeric liver-specific promoters (LSPs), a cytomegalovirusenhancer/chicken beta-actin (CAG) promoter, a tetracycline responsivepromoter (TRE), a transthyretin promoter (TTR), an simian virus 40(SV40) promoter and a CK6 promoter. The sequences of these and numerousadditional promoters are known in the art. The relevant sequences may bereadily obtained from public databases and incorporated into vectors foruse in practicing the present invention.

The present invention also contemplates the inclusion of a generegulation system for the controlled expression of the coding sequencefor two or more polypeptides or proteins of interest. Gene regulationsystems are useful in the modulated expression of a particular gene orgenes. In one exemplary approach, a gene regulation system or switchincludes a chimeric transcription factor that has a ligand bindingdomain, a transcriptional activation domain and a DNA binding domain.The domains may be obtained from virtually any source and may becombined in any of a number of ways to obtain a novel protein. Aregulatable gene system also includes a DNA response element whichinteracts with the chimeric transcription factor. This element islocated adjacent to the gene to be regulated.

Exemplary gene regulation systems that may be employed in practicing thepresent invention include, the Drosophila ecdysone system (Yao et al.,Proc. Nat. Acad. Sci., 93:3346 (1996)), the Bombyx ecdysone system (Suhret al., Proc. Nat. Acad. Sci., 95:7999 (1998)), the Valentis GeneSwitch®synthetic progesterone receptor system which employs RU486 as theinducer (Osterwalder et al., Proc Natl Acad Sci 98(22):12596-601(2001)); the Tet™ & RevTet™ Systems (BD Biosciences Clontech), whichemploys small molecules, such as tetracycline (Tc) or analogues, e.g.doxycycline, to regulate (turn on or off) transcription of the target(Knott et al., Biotechniques 32(4):796, 798, 800 (2002)); ARIADRegulation Technology which is based on the use of a small molecule tobring together two intracellular molecules, each of which is linked toeither a transcriptional activator or a DNA binding protein. When thesecomponents come together, transcription of the gene of interest isactivated. Ariad has two major systems: a system based onhomodimerization and a system based on heterodimerization (Rivera etal., Nature Med, 2(9):1028-1032 (1996); Ye et al., Science 283: 88-91(2000)), either of which may be incorporated into the vectors of thepresent invention.

Preferred gene regulation systems for use in practicing the presentinvention are the ARIAD Regulation Technology and the Tet™ & RevTet™Systems.

C. Non-Viral Vectors

Several non-viral methods for the transfer of expression vectors intocells also are contemplated by the present invention. These includecalcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen andOkayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985),electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), directmicroinjection (Harland and Weintraub, 1985), DNA-loaded liposomes(Nicolau and Sene, 1982; Fraley et al., 1979) and liofectamine-DNAcomplex, cell sonication (Fechheimer et al., 1987), gene bombardmentusing high velocity microprojectiles (Yang et al., 1990), polycations(Bousssif et al., 1995) and receptor-mediated transfection (Wu and Wu,1987; Wu and Wu, 1988). Some of these techniques may be successfullyadapted for in vivo or ex vivo use. A person of ordinary skill in theart would be familiar with the techniques pertaining to use of nonviralvectors, and would understand that other types of nonviral vectors thanthose disclosed herein are contemplated by the present invention.

In a further embodiment of the invention, the expression cassette may beentrapped in a liposome or lipid formulation. Liposomes are vesicularstructures characterized by a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated is a gene construct complexed withLipofectamine (Gibco BRL). One of ordinary skill in the art would befamiliar with techniques utilizing liposomes and lipid formulations.

Lipid based non-viral formulations provide an alternative to adenoviralgene therapies. Although many cell culture studies have documented lipidbased non-viral gene transfer, systemic gene delivery via lipid basedformulations has been limited. A major limitation of non-viral lipidbased gene delivery is the toxicity of the cationic lipids that comprisethe non-viral delivery vehicle. The in vivo toxicity of liposomespartially explains the discrepancy between in vitro and in vivo genetransfer results. Another factor contributing to this contradictory datais the difference in liposome stability in the presence and absence ofserum proteins. The interaction between liposomes and serum proteins hasa dramatic impact on the stability characteristics of liposomes (Yangand Huang, 1997). Cationic liposomes attract and bind negatively chargedserum proteins. Liposomes coated by serum proteins are either dissolvedor taken up by macrophages leading to their removal from circulation.Current in vivo liposomal delivery methods use subcutaneous,intradermal, or intracranial injection to avoid the toxicity andstability problems associated with cationic lipids in the circulation.The interaction of liposomes and plasma proteins is responsible for thedisparity between the efficiency of in vitro (Felgner et al., 1987) andin vivo gene transfer (Zhu et al., 1993; Solodin et al., 1995; Thierryet al., 1995; Tsukamoto et al., 1995; Aksentijevich et al., 1996).

The production of lipid formulations often is accomplished by sonicationor serial extrusion of liposomal mixtures after (I) reverse phaseevaporation (II) dehydration-rehydration (III) detergent dialysis and(IV) thin film hydration. Once manufactured, lipid structures can beused to encapsulate compounds that are toxic (chemotherapeutics) orlabile (nucleic acids) when in circulation. Liposomal encapsulation hasresulted in a lower toxicity and a longer serum half-life for suchcompounds (Gabizon et al., 1990). Numerous disease treatments are usinglipid based gene transfer strategies to enhance conventional orestablish novel therapies, in particular therapies for treatinghyperproliferative diseases.

D. Delivery of Nucleic Acid Constructs Including Protein or PolypeptideCoding Sequences to Cells

The vector constructs that may be employed by the invention comprisingnucleic acid sequences encoding heterologous proteins or polypeptides,and a self-processing cleavage site alone or in combination with asequence encoding an additional proteolytic cleavage site may beintroduced into cells in vitro, ex vivo or in vivo for expression ofheterologous coding sequences by cells, e.g., somatic cells in vivo, orfor the production of recombinant polypeptides by vector-transducedcells, in vitro or in vivo.

The vector constructs of the invention may be introduced into cells invitro or ex vivo using standard methodology known in the art. Suchtechniques include transfection using calcium phosphate, microinjectioninto cultured cells (Capecchi, Cell 22:479-488 (1980)), electroporation(Shigekawa et al., BioTechn., 6:742-751 (1988)), liposome-mediated genetransfer (Mannino et al., BioTechn., 6:682-690 (1988)), lipid-mediatedtransduction (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417(1987)), and nucleic acid delivery using high-velocity microprojectiles(Klein et al., Nature 327:70-73 (1987)).

For in vitro or ex vivo expression, any cell effective to express afunctional protein may be employed. Numerous examples of cells and celllines used for protein expression are known in the art. For example,prokaryotic cells and insect cells may be used for expression. Inaddition, eukaryotic microorganisms, such as yeast may be used. Theexpression of recombinant proteins in prokaryotc, insect and yeastsystems are generally known in the art and may be adapted for protein orpolypeptide expression using the compositions and methods of the presentinvention.

Exemplary host cells useful for expression further include mammaliancells, such as fibroblast cells, cells from non-human mammals such asovine, porcine, murine and bovine cells, insect cells and the like.Specific examples of mammalian cells include COS cells, VERO cells, HeLacells, Chinese hamster ovary (CHO) cells, 293 cell, NSO cells, 3T3fibroblast cells, W138 cells, BHK cells, HEPG2 cells, DUX cells and MDCKcells.

Host cells are cultured in conventional nutrient media, modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. Mammalian hostcells may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), Sigma),RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma)are typically suitable for culturing host cells. A given medium isgenerally supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleosides (such as adenosine and thymidine),antibiotics, trace elements, and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theappropriate culture conditions for a particular cell line, such astemperature, pH and the like, are generally known in the art, withsuggested culture conditions for culture of numerous cell lines forexample in the ATCC Catalogue available on line at“http://www.atcc.org/SearchCatalogs/AllCollections.cfm”

The vectors of the invention may be administered in vivo via variousroutes (e.g., intradermally, intravenously, intratumorally, into thebrain, intraportally, intraperitoneally, intramuscularly, into thebladder etc.), to deliver multiple genes connected via a self processingcleavage sequence to express two or more proteins or polypeptides inanimal models or human subjects. Dependent upon the route ofadministration, the therapeutic proteins elicit their effect locally(e.g., in brain or bladder) or systemically (other routes ofadministration). The use of tissue specific promoters 5′ to the openreading frame(s) for a protein or polypeptide in the vectors of theinvention may be used to effect tissue specific expression of the two ormore proteins or polypeptides encoded by the vector.

Various methods that introduce a recombinant vector carrying a transgeneinto target cells in vitro, ex vivo or in vivo have been previouslydescribed and are well known in the art. The present invention providesfor therapeutic methods, vaccines, and cancer therapies by transducingtarget cells with recombinant vectors of the invention.

For example, in vivo delivery of the recombinant vectors of theinvention may be targeted to a wide variety of organ types including,but not limited to brain, liver, blood vessels, muscle, heart, lung andskin.

In the case of ex vivo gene transfer, the target cells are removed fromthe host and genetically modified in the laboratory using recombinantvectors of the present invention and methods well known in the art.

The recombinant vectors of the invention can be administered usingconventional modes of administration including but not limited to themodes described above. The recombinant vectors of the invention may beprovided in any of a variety of formulations such as liquid solutionsand suspensions, microvesicles, liposomes and injectable or infusiblesolutions. The preferred form depends upon the mode of administrationand the therapeutic application. A from appropriate to the route ofdelivery may be readily determined using knowledge generally availableto those of skill in the relevant art.

The many advantages to be realized in using the inventive recombinantvector constructs of the invention in recombinant protein andpolypeptide production in vivo include administration of a single vectorfor long-term and sustained expression of two or more recombinantprotein or polypeptide ORFs in patients; in vivo expression of two ormore recombinant protein or polypeptide ORFs having biological activity;and the natural posttranslational modifications of the recombinantprotein or polypeptide generated in human cells.

One preferred aspect is use of the recombinant vector constructs of thepresent invention for the in vitro production of recombinant proteinsand polypeptides. Methods for recombinant protein production are wellknown in the art and self processing cleavage site-containing vectorconstructs of the present invention may be utilized for expression ofrecombinant proteins and polypeptides using such standard methodology.

In one exemplary aspect of the invention, vector introduction oradministration to a cell (transfection) is followed by one or more ofthe following steps:

-   -   (1) culturing the transfected cell under conditions to selecting        for a cell expressing the recombinant protein or polypeptide;    -   (2) evaluating expression of the recombinant protein or        polypeptide; and    -   (3) collecting the recombinant protein or polypeptide.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andbeta.-actin promoters). Other viral promoters which may be employedinclude, but are not limited to, adenovirus promoters, thymidine kinase(TK) promoters, and B19 parvovirus promoters. The selection of asuitable promoter will be apparent to those skilled in the art from theteachings contained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, Ψ-2,Ψ-AM, PA12, T19-14X, VT-19-17-H2, ΨCRE, . ΨCRIP, GP+E-86, GP+envAm12,and DAN cell lines as described in Miller, Human Gene Therapy, Vol. 1,pgs. 5-14 (1990), which is incorporated herein by reference in itsentirety. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

IV. Host Cells Containing an Exogenously Supplied Coding Nucleic AcidMolecule

The present invention further provides host cells transformed with anucleic acid molecule that encodes an renalase protein. The host cellcan be either prokaryotic or eukaryotic. Eukaryotic cells useful forexpression of a protein of the invention are not limited, so long as thecell line is compatible with cell culture methods and compatible withthe propagation of the expression vector and expression of the geneproduct. Preferred eukaryotic host cells include, but are not limitedto, yeast, insect and mammalian cells, preferably vertebrate cells suchas those from a mouse, rat, monkey or human cell line, and otherimmortalized cell lines. Preferred eukaryotic host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, babyhamster kidney cells (BHK), and the like eukaryotic tissue culture celllines, and other immortalized cell line.

Any prokaryotic host can be used to express a rDNA molecule encoding aprotein of the invention. The preferred prokaryotic host is E. coli.

Transformation of appropriate cell hosts with a rDNA molecule of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used and host system employed. With regardto transformation of prokaryotic host cells, electroporation and salttreatment methods are typically employed, see, for example, Cohen etal., Proc. Natl. Acad. Sci. USA 69:2110, 1972; and Maniatis et al.,Molecular Cloning, A Laboratory Mammal, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982). With regard to transformation ofvertebrate cells with vectors containing rDNAs, electroporation,cationic lipid or salt treatment methods are typically employed, see,for example, Graham et al., Virol. 52:456, 1973; Wigler et al., Proc.Natl. Acad. Sci. USA 76:1373-76, 1979.

Successfully transformed cells, i.e., cells that contain a rDNA moleculeof the present invention, can be identified by well known techniquesincluding the selection for a selectable marker. For example, cellsresulting from the introduction of an rDNA of the present invention canbe cloned to produce single colonies. Cells from those colonies can beharvested, lysed and their DNA content examined for the presence of therDNA using a method such as that described by Southern, J. Mol. Biol.98:503, 1975, or Berent et al., Biotech. 3:208, 1985 or the proteinsproduced from the cell assayed via an immunological method.

V. Antisense Molecules, Ribozymes, and Interfering RNA

Further, the invention includes a recombinant cell comprising anantisense nucleic acid which cell is a useful model for elucidating therole(s) of renalase in cellular processes. That is, the increasedexpression of renalase in balloon-injured vessels and, morespecifically, in the adventitia thereof, indicate that renalase isinvolved in cell proliferation associated with negative remodeling andarterial restenosis. Accordingly, a transgenic cell comprising anantisense nucleic acid complementary to renalase but in an antisenseorientation is a useful tool for the study of the mechanism(s) of actionof renalase and its role(s) in the cell and for the identification oftherapeutics that ameliorate the effect(s) of renalase expression.

One skilled in the art can appreciate, based upon the disclosureprovided herein, that an antisense nucleic acid complementary to anucleic acid encoding renalase can be used to transfect a cell and thecell can be studied to determine the effect(s) of altered expression ofrenalase in order to study the function(s) of renalase and to identityuseful therapeutics and diagnostics.

Further, methods of decreasing renalase expression and/or activity in acell can provide useful diagnostics and/or therapeutics for diseases,disorders or conditions mediated by or associated with increasedrenalase expression, increased level of renalase protein in a cell orsecretion therefrom, and/or increased renalase activity.

One skilled in the art will appreciate that one way to decrease thelevels of renalase mRNA and/or protein in a cell is to inhibitexpression of the nucleic acid encoding the protein. Expression ofrenalase may be inhibited using, for example, antisense molecules, andalso by using ribozymes or double-stranded RNA as described in, forexample, Wianny and Kemicka-Goetz (2000, Nature Cell Biol. 2:70-75).

RNA interference (RNAi) is a phenomenon in which the introduction ofdouble-stranded RNA (dsRNA) into a diverse range of organisms and celltypes causes degradation of the complementary mRNA. In the cell, longdsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs,or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequentlyassemble with protein components into an RNA-induced silencing complex(RISC), unwinding in the process. Activated RISC then binds tocomplementary transcript by base pairing interactions between the siRNAantisense strand and the mRNA. The bound mRNA is cleaved and sequencespecific degradation of mRNA results in gene silencing. See, forexample, U.S. Pat. No. 6,506,559; Fire et al., Nature (1998)391(19):306-311; Timmons et al., Nature (1998) 395:854; Montgomery etal., TIG (1998) 14(7):255-258; David R. Engelke, Ed., RNA Interference(RNAi) Nuts & Bolts of RNAi Technology, DNA Press (2003); and Gregory J.Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring HarborLaboratory Press (2003). Therefore, the present invention also includesmethods of silencing the gene encoding renalase by using RNAitechnology.

Antisense molecules and their use for inhibiting gene expression arewell known in the art (see, e.g., Cohen, 1989, In:Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRCPress). Antisense nucleic acids are DNA or RNA molecules that arecomplementary, as that term is defined elsewhere herein, to at least aportion of a specific mRNA molecule (Weintraub, 1990, ScientificAmerican 262:40). In the cell, antisense nucleic acids hybridize to thecorresponding mRNA, forming a double-stranded molecule therebyinhibiting the translation of genes.

The use of antisense methods to inhibit the translation of genes isknown in the art, and is described, for example, in Marcus-Sakura (1988,Anal. Biochem. 172:289). Such antisense molecules may be provided to thecell via genetic expression using DNA encoding the antisense molecule astaught by Inoue (1993, U.S. Pat. No. 5,190,931).

Alternatively, antisense molecules of the invention may be madesynthetically and then provided to the cell. Antisense oligomers ofbetween about 10 to about 30, and more preferably about 15 nucleotides,are preferred, since they are easily synthesized and introduced into atarget cell. Synthetic antisense molecules contemplated by the inventioninclude oligonucleotide derivatives known in the art which have improvedbiological activity compared to unmodified oligonucleotides (see Cohen,supra; Tullis, 1991, U.S. Pat. No. 5,023,243, incorporated by referenceherein in its entirety).

Ribozymes and their use for inhibiting gene expression are also wellknown in the art (see, e.g., Cech et al., 1992, J. Biol. Chem.267:17479-17482; Hampel et al., 1989, Biochemistry 28:4929-4933;Eckstein et al., International Publication No. WO 92/07065; Altman etal., U.S. Pat. No. 5,168,053, incorporated by reference herein in itsentirety). Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. Through the modification of nucleotidesequences encoding these RNAs, molecules can be engineered to recognizespecific nucleotide sequences in an RNA molecule and cleave it (Cech,1988, J. Amer. Med. Assn. 260:3030). A major advantage of this approachis that, because they are sequence-specific, only mRNAs with particularsequences are inactivated.

There are two basic types of ribozymes, namely, tetrahymena-type(Hasselhoff, 1988, Nature 334:585) and hammerhead-type. Tetrahymena-typeribozymes recognize sequences which are four bases in length, whilehammerhead-type ribozymes recognize base sequences 11-18 bases inlength. The longer the sequence, the greater the likelihood that thesequence will occur exclusively in the target mRNA species.Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating specific mRNA species, and18-base recognition sequences are preferable to shorter recognitionsequences which may occur randomly within various unrelated mRNAmolecules.

Ribozymes useful for inhibiting the expression of renalase can bedesigned by incorporating target sequences into the basic ribozymestructure which are complementary to the mRNA sequence of the renalaseencoded by renalase or having at least about 33% homology to SEQ IDNO:1. Preferably, the sequence is at least about 35% homologous, evenmore preferably, at least about 40% homologous, even more preferably, atleast about 45% homologous, yet more preferably, at least about 50%homologous, more preferably, at least about 55% homologous, morepreferably, at least about 60% homologous, even more preferably, atleast about 65% homologous, yet more preferably, at least about 70%homologous, more preferably, at least about 75% homologous, even morepreferably, at least about 80% homologous, yet more preferably, at leastabout 85% homologous, more preferably, at least about 90% homologous,even more preferably, at least about 95% homologous, and mostpreferably, at least about 99% homologous to SEQ ID NO: 1. Ribozymestargeting renalase may be synthesized using commercially availablereagents (Applied Biosystems, Inc., Foster City, Calif.) or they may begenetically expressed from DNA encoding them.

V. Recombinant Cells and Transgenic Non-Human Mammals

The invention includes a recombinant cell comprising, inter alia, anisolated nucleic acid encoding renalase, an antisense nucleic acidcomplementary thereto, a nucleic acid encoding an antibody thatspecifically binds renalase, and the like. In one aspect, therecombinant cell can be transiently transfected with a vector (e.g., aplasmid, and the like) encoding a portion of the nucleic acid encodingrenalase. The nucleic acid need not be integrated into the cell genomenor does it need to be expressed in the cell. Moreover, the cell may bea prokaryotic or a eukaryotic cell and the invention should not beconstrued to be limited to any particular cell line or cell type. Suchcells include, but are not limited to, fibroblasts, mouse stem cells,amphibian oocytes, osteoblasts, smooth muscle cells, endothelial cells,and the like.

In one aspect, the recombinant cell comprising an isolated nucleic acidencoding mammalian renalase is used to produce a transgenic non-humanmammal. That is, the exogenous nucleic acid, or “transgene” as it isalso referred to herein, of the invention is introduced into a cell, andthe cell is then used to generate the non-human transgenic mammal. Thecell into which the transgene is introduced is preferably an embryonicstem (ES) cell. However, the invention should not be construed to belimited solely to ES cells comprising the transgene of the invention norto cells used to produce transgenic animals. Rather, a transgenic cellof the invention includes, but is not limited to, any cell derived froma transgenic animal comprising a transgene, a cell comprising thetransgene derived from a chimeric animal derived from the transgenic EScell, and any other comprising the transgene which may or may not beused to generate a non-human transgenic mammal.

Further, it is important to note that the purpose oftransgene-comprising, i.e., recombinant, cells should not be construedto be limited to the generation of transgenic mammals. Rather, theinvention should be construed to include any cell type into which anucleic acid encoding a mammalian renalase is introduced, including,without limitation, a prokaryotic cell and a eukaryotic cell comprisingan isolated nucleic acid encoding mammalian renalase.

When the cell is a eukaryotic cell, the cell may be any eukaryotic cellwhich, when the transgene of the invention is introduced therein, andthe protein encoded by the desired gene is no longer expressedtherefrom, a benefit is obtained. Such a benefit may include the factthat there has been provided a system in which lack of expression of thedesired gene can be studied in vitro in the laboratory or in a mammal inwhich the cell resides, a system wherein cells comprising the introducedgene deletion can be used as research, diagnostic and therapeutic tools,and a system wherein animal models are generated which are useful forthe development of new diagnostic and therapeutic tools for selecteddisease states in a mammal including, for example, ESRD andhypertension.

Alternatively, the invention includes a eukaryotic cell which, when thetransgene of the invention is introduced therein, and the proteinencoded by the desired gene is expressed therefrom where it was notpreviously present or expressed in the cell or where it is now expressedat a level or under circumstances different than that before thetransgene was introduced, a benefit is obtained. Such a benefit mayinclude the fact that there has been provided a system in the expressionof the desired gene can be studied in vitro in the laboratory or in amammal in which the cell resides, a system wherein cells comprising theintroduced gene can be used as research, diagnostic and therapeutictools, and a system wherein animal models are generated which are usefulfor the development of new diagnostic and therapeutic tools for selecteddisease states in a mammal.

Such cell expressing an isolated nucleic acid encoding renalase can beused to provide renalase to a cell, tissue, or whole animal where ahigher level of renalase can be useful to treat or alleviate a disease,disorder or condition associated with low level of renalase expressionand/or activity. Such diseases, disorders or conditions can include, butare not limited to, ESRD, hypertension, cardiovascular diseases.Additional expression of renalase could thus lead to lower bloodpressure. Therefore, the invention includes a cell expressing renalaseto increase or induce renalase expression, translation, and/or activity,where increasing renalase expression, protein level, and/or activity canbe useful to treat or alleviate a disease, disorder or condition.

One of ordinary skill would appreciate, based upon the disclosureprovided herein, that a “knock-in” or “knock-out” vector of theinvention comprises at least two sequences homologous to two portions ofthe nucleic acid which is to be replaced or deleted, respectively. Thetwo sequences are homologous with sequences that flank the gene; thatis, one sequence is homologous with a region at or near the 5′ portionof the coding sequence of the nucleic acid encoding renalase and theother sequence is further downstream from the first. One skilled in theart would appreciate, based upon the disclosure provided herein, thatthe present invention is not limited to any specific flanking nucleicacid sequences. Instead, the targeting vector may comprise two sequenceswhich remove some or all (i.e., a “knock-out” vector) or which insert(i.e., a “knock-in” vector) a nucleic acid encoding renalase, or afragment thereof, from or into a mammalian genome, respectively. Thecrucial feature of the targeting vector is that it comprise sufficientportions of two sequences located towards opposite, i.e., 5′ and 3′,ends of the renalase open reading frame (ORF) in the case of a“knock-out” vector, to allow deletion/insertion by homologousrecombination to occur such that all or a portion of the nucleic acidencoding renalase is deleted from or inserted into a location on amammalian chromosome.

The design of transgenes and knock-in and knock-out targeting vectors iswell-known in the art and is described in standard treatises such asSambrook et al. (1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York), and in Ausubel et al. (1997,Current Protocols in Molecular Biology, John Wiley & Sons, New York),and the like. The upstream and downstream portions flanking or withinthe renalase coding region to be used in the targeting vector may beeasily selected based upon known methods and following the teachingsdisclosed herein based on the disclosure provided herein including thenucleic and amino acid sequences of both rat and human renalase. Armedwith these sequences, one of ordinary skill in the art would be able toconstruct the transgenes and knock-out vectors of the invention.

The invention further includes a knock-out targeting vector comprising anucleic acid encoding a selectable marker such as, for example, anucleic acid encoding the neo^(R) gene thereby allowing the selection oftransgenic a cell where the nucleic acid encoding renalase, or a portionthereof, has been deleted and replaced with the neomycin resistance geneby the cell's ability to grow in the presence of G418. However, thepresent invention should not be construed to be limited to neomycinresistance as a selectable marker. Rather, other selectable markerswell-known in the art may be used in the knock-out targeting vector toallow selection of recombinant cells where the renalase gene has beendeleted and/or inactivated and replaced by the nucleic acid encoding theselectable marker of choice. Methods of selecting and incorporating aselectable marker into a vector are well-known in the art and aredescribe in, for example, Sambrook et al. (1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inAusubel et al. (1997, Current Protocols in Molecular Biology, John Wiley& Sons, New York).

As noted herein, the invention includes a non-human transgenic mammalcomprising an exogenous nucleic acid inserted into a desired site in thegenome thereof thereby deleting the coding region of a desiredendogenous target gene, i.e., a knock-out transgenic mammal. Further,the invention includes a transgenic non-human mammal wherein anexogenous nucleic acid encoding renalase is inserted into a site thegenome, i.e., a “knock-in” transgenic mammal. The knock-in transgeneinserted may comprise various nucleic acids encoding, for example, a tagpolypeptide, a promoter/regulatory region operably linked to the nucleicacid encoding renalase not normally present in the cell or not typicallyoperably linked to renalase.

The generation of the non-human transgenic mammal of the invention ispreferably accomplished using the method which is now described.However, the invention should in no way be construed as being limitedsolely to the use of this method, in that, other methods can be used togenerate the desired knock-out mammal. In the preferred method ofgenerating a non-human transgenic mammal, ES cells are generatedcomprising the transgene of the invention and the cells are then used togenerate the knock-out animal essentially as described in Nagy andRossant (1993, In: Gene Targeting, A Practical Approach, pp. 146-179,Joyner ed., IRL Press). ES cells behave as normal embryonic cells ifthey are returned to the embryonic environment by injection into a hostblastocyst or aggregate with blastomere stage embryos. When so returned,the cells have the full potential to develop along all lineages of theembryo. Thus, it is possible, to obtain ES cells, introduce a desiredDNA therein, and then return the cell to the embryonic environment fordevelopment into mature mammalian cells, wherein the desired DNA may beexpressed.

Precise protocols for the generation of transgenic mice are disclosed inNagy and Rossant (1993, In: Gene Targeting, A Practical Approach, Joynered. IRL Press, pp. 146-179). and are therefore not repeated herein.Transfection or transduction of ES cells in order to introduce thedesired DNA therein is accomplished using standard protocols, such asthose described, for example, in Sambrook et al. (1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York),and in Ausubel et al. (1997, Current Protocols in Molecular Biology,John Wiley & Sons, New York). Preferably, the desired DNA containedwithin the transgene of the invention is electroporated into ES cells,and the cells are propagated as described in Soriano et al. (1991, Cell64:693-702).

Introduction of an isolated nucleic acid into the fertilized egg of themammal is accomplished by any number of standard techniques intransgenic technology (Hogan et al., 1986, Manipulating the MouseEmbryo: A Laboratory Manual, Cold Spring Harbor, NY). Most commonly, thenucleic acid is introduced into the embryo by way of microinjection.

Once the nucleic acid is introduced into the egg, the egg is incubatedfor a short period of time and is then transferred into a pseudopregnantmammal of the same species from which the egg was obtained as described,for example, in Hogan et al. (1986, Manipulating the Mouse Embryo: ALaboratory Manual, Cold Spring Harbor, N.Y.). Typically, many eggs areinjected per experiment, and approximately two-thirds of the eggssurvive the procedure. About twenty viable eggs are then transferredinto pseudopregnant animals, and usually four to ten of the viable eggsso transferred will develop into live pups.

Any mammalian renalase gene may be used in the methods described hereinto produce a transgenic mammal or a transgenic cell harboring atransgene comprising a deletion of all or part of that mammalianrenalase gene.

The transgenic mammal of the invention can be any species of mammal.Thus, the invention should be construed to include generation oftransgenic mammals encoding the chimeric nucleic acid, which mammalsinclude mice, hamsters, rats, rabbits, pigs, sheep and cattle. Themethods described herein for generation of transgenic mice can beanalogously applied using any mammalian species. Preferably, thetransgenic mammal of the invention is a rodent and even more preferably,the transgenic mammal of the invention is a mouse. By way of example,Lukkarinen et al. (1997, Stroke 28:639-645), teaches that geneconstructs which enable the generation of transgenic mice also enablethe generation of other transgenic rodents, including rats. Similarly,nullizygous mutations in a genetic locus of an animal of one species canbe replicated in an animal of another species having a genetic locushighly homologous to the first species.

To identify the transgenic mammals of the invention, pups are examinedfor the presence of the isolated nucleic acid using standard technologysuch as Southern blot hybridization, PCR, and/or RT-PCR. Expression ofthe nucleic acid in the cells and in the tissues of the mammal is alsoassessed using ordinary technology described herein. Further, thepresence or absence of renalase in the circulating blood of thetransgenic animal can be determined, if the protein is secreted, byusing, for example, Western blot analysis, or using standard methods forprotein detection that are well-known in the art.

Cells obtained from the transgenic mammal of the invention, which arealso considered “transgenic cells” as the term is used herein, encompasssuch as cells as those obtained from the renalase (+/−) and (−/−)transgenic non-human mammal described elsewhere herein, are usefulsystems for modeling diseases and symptoms of mammals which are believedto be associated with altered levels of renalase expression such asESRD, hypertension, cardiovascular diseases. and any other disease,disorder or condition associated with an altered level of renalaseexpression.

Particularly suitable are cells derived from a tissue of the non-humanknock-out or knock-in transgenic mammal described herein, wherein thetransgene comprising the renalase gene is expressed or inhibitsexpression of renalase in various tissues. By way of example, cell typesfrom which such cells are derived include fibroblasts and like cells of(1) the renalase (+/+), (+/−) and (−/−) non-human transgenic livebornmammal, (2) the renalase (+/+), (−/−) or (+/−) fetal animal, and (3)placental cell lines obtained from the renalase (+/+), (−/−) and (+/−)fetus and liveborn mammal.

One skilled in the art would appreciate, based upon this disclosure,that cells comprising decreased levels of renalase protein, decreasedlevel of renalase activity, or both, include, but are not limited to,cells expressing inhibitors of renalase expression (e.g., antisense orribozyme molecules).

Methods and compositions useful for maintaining mammalian cells inculture are well known in the art, wherein the mammalian cells areobtained from a mammal including, but not limited to, cells obtainedfrom a mouse such as the transgenic mouse described herein.

Alternatively, recombinant cells expressing renalase can be administeredin ex vivo and in vivo therapies where administering the recombinantcells thereby administers the protein to a cell, a tissue, and/or ananimal. Additionally, the recombinant cells are useful for the discoveryof renalase ligand(s) and renalase signaling pathway(s).

The recombinant cell of the invention may be used to study the effectsof elevated or decreased renalase levels on cell homeostasis and cellproliferation and/or migration since renalase has been hypothesized toplay a role in cell migration, adventitial fibrosis, arterialrestenosis, negative remodeling, and the like

The recombinant cell of the invention, wherein the cell has beenengineered such that it does not express renalase, or expresses reducedor altered renalase lacking biological activity, can also be used in exvivo and in vivo cell therapies where either an animal's own cells(e.g., fibroblasts, and the like), or those of a syngeneic matcheddonor, are recombinantly engineered as described elsewhere herein (e.g.,by insertion of an antisense nucleic acid or a knock-out vector suchthat renalase expression and/or protein levels are thereby reduced inthe recombinant cell), and the recombinant cell is administered to therecipient animal. In this way, recombinant cells that express renalaseat a reduced level can be administered to an animal whose own cellsexpress increased levels of renalase thereby treating or alleviating adisease, disorder or condition associated with or mediated by increasedrenalase expression as disclosed elsewhere herein.

The transgenic mammal of the invention, rendered susceptible toadventitial fibrosis, arterial restenosis, and the like, such as, forexample, a renalase knock-out mouse, can be used to study thepathogenesis of these diseases and the potential role of renalasetherein.

Further, the transgenic mammal and/or cell of the invention may be usedto further study the subcellular localization of renalase. Also, thetransgenic mammal (both +/− and −/− live born and fetuses) and/or cellof the invention may be used to study to role(s) of renalase incatecholamine circulation to elucidate the target(s) of renalase actionas well as any receptor(s) and/or ligands that bind with renalase tomediate its effect(s) in the cell.

VI. Antibodies

The invention also includes an antibody that specifically bindsrenalase, or a fragment thereof. One skilled in the art wouldunderstand, based upon the disclosure provided herein, that an antibodythat specifically binds renalase, binds with a protein of the invention,such as, but not limited to human renalase or an immunogenic portionthereof. In one embodiment, the antibody is directed to rat renalasecomprising the amino acid sequence of SEQ ID NO:2.

Polyclonal antibodies are generated by immunizing rabbits according tostandard immunological techniques well-known in the art (see, e.g.,Harlow et al., 1988, In: Antibodies, A Laboratory Manual, Cold SpringHarbor, N.Y.). Such techniques include immunizing an animal with achimeric protein comprising a portion of another protein such as amaltose binding protein or glutathione (GSH) tag polypeptide portion,and/or a moiety such that the renalase portion is rendered immunogenic(e.g., renalase conjugated with keyhole limpet hemocyanin, KLH) and aportion comprising the respective rodent and/or human renalase aminoacid residues. The chimeric proteins are produced by cloning theappropriate nucleic acids encoding renalase (e.g., SEQ ID NO: 1) into aplasmid vector suitable for this purpose, such as but not limited to,pMAL-2 or pCMX.

However, the invention should not be construed as being limited solelyto these antibodies or to these portions of the protein antigens.Rather, the invention should be construed to include other antibodies,as that term is defined elsewhere herein, to rat and human renalase, orportions thereof. Further, the present invention should be construed toencompass antibodies, inter alia, bind with renalase and they are ableto bind renalase present on Western blots, in immunohistochemicalstaining of tissues thereby localizing renalase in the tissues, and inimmunofluorescence microscopy of a cell transiently transfected with anucleic acid encoding at least a portion of renalase.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the antibody can specifically bind with anyportion of the protein and the full-length protein can be used togenerate antibodies specific therefor. However, the present invention isnot limited to using the full-length protein as an immunogen. Rather,the present invention includes using an immunogenic portion of theprotein to produce an antibody that specifically binds with mammalianrenalase. That is, the invention includes immunizing an animal using animmunogenic portion, or antigenic determinant, of the renalase protein.The antibodies can be produced by immunizing an animal such as, but notlimited to, a rabbit or a mouse, with a protein of the invention, or aportion thereof, or by immunizing an animal using a protein comprisingat least a portion of renalase, or a fusion protein including a tagpolypeptide portion comprising, for example, a maltose binding proteintag polypeptide portion covalently linked with a portion comprising theappropriate renalase amino acid residues. One skilled in the art wouldappreciate, based upon the disclosure provided herein, that smallerfragments of these proteins can also be used to produce antibodies thatspecifically bind renalase.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that various portions of an isolated renalasepolypeptide can be used to generate antibodies to either highlyconserved regions of renalase or to non-conserved regions of thepolypeptide. As disclosed elsewhere herein, renalase comprises variousconserved domains including, but not limited to, a putative signalpeptide from at the N terminus, a FAD binding domain (amino acidresidues from about 4 to 35); an amine oxidase domain (amino acidresidues from about 75 to 339).

Once armed with the sequence of renalase and the detailed analysislocalizing the various conserved and non-conserved domains of theprotein, the skilled artisan would understand, based upon the disclosureprovided herein, how to obtain antibodies specific for the variousportions of a mammalian renalase polypeptide using methods well-known inthe art or to be developed, as well as methods disclosed herein.

Further, the skilled artisan, based upon the disclosure provided herein,would appreciate that the non-conserved regions of a protein of interestcan be more immunogenic than the highly conserved regions which areconserved among various organisms. Further, immunization using anon-conserved immunogenic portion can produce antibodies specific forthe non-conserved region thereby producing antibodies that do notcross-react with other proteins which can share one or more conservedportions. Thus, one skilled in the art would appreciate, based upon thedisclosure provided herein, that the non-conserved regions of eachrenalase molecule can be used to produce antibodies that are specificonly for that renalase and do not cross-react non-specifically withother renalases or with other proteins.

Alternatively, the skilled artisan would also understand, based upon thedisclosure provided herein, that antibodies developed using a regionthat is conserved among one or more renalase molecule can be used toproduce antibodies that react specifically with one or more renalasemolecule. Methods for producing antibodies that specifically bind with aconserved protein domain which may otherwise be less immunogenic thanother portions of the protein are well-known in the art and include, butare not limited to, conjugating the protein fragment of interest to amolecule (e.g., keyhole limpet hemocyanin, and the like), therebyrendering the protein domain immunogenic, or by the use of adjuvants(e.g., Freund's complete and/or incomplete adjuvant, and the like), orboth. Thus, the invention encompasses antibodies that recognize at leastone renalase and antibodies that specifically bind with more than onerenalase, including antibodies that specifically bind with all renalase.

One skilled in the art would appreciate, based upon the disclosureprovided herein, which portions of renalase are less homologous withother proteins sharing conserved domains. However, the present inventionis not limited to any particular domain; instead, the skilled artisanwould understand that other non-conserved regions of the renalaseproteins of the invention can be used to produce the antibodies of theinvention as disclosed herein.

Therefore, the skilled artisan would appreciate, based upon thedisclosure provided herein, that the present invention encompassesantibodies that neutralize and/or inhibit renalase activity (e.g., byinhibiting necessary renalase receptor/ligand interactions) whichantibodies can recognize one or more renalases, including, but notlimited to human renalase, as well as renalases from various species(e.g., mouse renalase).

The invention should not be construed as being limited solely to theantibodies disclosed herein or to any particular immunogenic portion ofthe proteins of the invention. Rather, the invention should be construedto include other antibodies, as that term is defined elsewhere herein,to renalase, or portions thereof, or to proteins sharing at least about15% homology with a polypeptide having the amino acid sequence of SEQ IDNO: 2. In other embodiments, the polypeptide is at least about 20%homologous, or at least about 25% homologous, or at least about 30%homologous, or at least about 35% homologous, or at least about 40%homologous, or at least about 45% homologous, or at least about 50%homologous, or at least about 55% homologous, or at least about 60%homologous, or at least about 65% homologous, or at least about 70%homologous, or at least about 75% homologous, or at least about 80%homologous, or at least about 85% homologous, or at least about 90%homologous, or at least about 95% homologous, or at least about 99%homologous to human renalase. In another embodiment, the polypeptidethat specifically binds with an antibody specific for mammalian renalaseis human renalase.

The invention encompasses polyclonal, monoclonal, synthetic antibodies,and the like. One skilled in the art would understand, based upon thedisclosure provided herein, that the crucial feature of the antibody ofthe invention is that the antibody bind specifically with renalase. Thatis, the antibody of the invention recognizes renalase, or a fragmentthereof (e.g., an immunogenic portion or antigenic determinant thereof),as demonstrated by antibody binding renalase on Western blots, inimmunostaining of cells, and/o immunoprecipitation of renalase, usingstandard methods well-known in the art.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the antibodies can be used to localize therelevant protein in a cell and to study the role(s) of the antigenrecognized thereby in cell processes. Moreover, the antibodies can beused to detect and or measure the amount of protein present in abiological sample using well-known methods such as, but not limited to,Western blotting and enzyme-linked immunosorbent assay (ELISA).Moreover, the antibodies can be used to immunoprecipitate and/orimmuno-affinity purify their cognate antigen using methods well-known inthe art.

In addition, the antibody can be used to decrease the level of renalasein a cell thereby inhibiting the effect(s) of renalase in a cell. Thus,by administering the antibody to a cell or to the tissues of an animalor to the animal itself, the required renalase receptor/ligandinteractions are therefore inhibited such that the effect ofrenalase-mediated signaling are also inhibited. One skilled in the artwould understand, based upon the disclosure provided herein, thatdetectable effects upon inhibiting renalase ligand/receptor interactionusing an anti-renalase antibody can include, but are not limited to,decreased proliferation of cells, decreased cell migration, decreasednegative modeling, decreased adventitial fibrosis, decreased arterialrestenosis, decreased fibrosis in any organ or tissue, decreasedossification or bone formation, and the like.

The generation of polyclonal antibodies is accomplished by inoculatingthe desired animal with the antigen and isolating antibodies whichspecifically bind the antigen therefrom using standard antibodyproduction methods such as those described in, for example, Harlow etal. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor,N.Y.).

Monoclonal antibodies directed against full length or peptide fragmentsof a protein or peptide may be prepared using any well known monoclonalantibody preparation procedures, such as those described, for example,in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold SpringHarbor, N.Y.) and in Tuszynski et al. (1988, Blood, 72:109-115).Quantities of the desired peptide may also be synthesized using chemicalsynthesis technology. Alternatively, DNA encoding the desired peptidemay be cloned and expressed from an appropriate promoter sequence incells suitable for the generation of large quantities of peptide.Monoclonal antibodies directed against the peptide are generated frommice immunized with the peptide using standard procedures as referencedherein.

Nucleic acid encoding the monoclonal antibody obtained using theprocedures described herein may be cloned and sequenced using technologywhich is available in the art, and is described, for example, in Wrightet al. (1992, Critical Rev. Immunol. 12:125-168), and the referencescited therein.

Further, the antibody of the invention may be “humanized” using thetechnology described in, for example, Wright et al. (supra), and in thereferences cited therein, and in Gu et al. (1997, Thrombosis andHematocyst 77:755-759), and other methods of humanizing antibodieswell-known in the art or to be developed.

To generate a phage antibody library, a cDNA library is first obtainedfrom mRNA which is isolated from cells, e.g., the hybridoma, whichexpress the desired protein to be expressed on the phage surface, e.g.,the desired antibody. cDNA copies of the mRNA are produced using reversetranscriptase. cDNA which specifies immunoglobulin fragments areobtained by PCR and the resulting DNA is cloned into a suitablebacteriophage vector to generate a bacteriophage DNA library comprisingDNA specifying immunoglobulin genes. The procedures for making abacteriophage library comprising heterologous DNA are well known in theart and are described, for example, in Sambrook et al., supra.

Bacteriophage which encode the desired antibody, may be engineered suchthat the protein is displayed on the surface thereof in such a mannerthat it is available for binding to its corresponding binding protein,e.g., the antigen against which the antibody is directed. Thus, whenbacteriophage which express a specific antibody are incubated in thepresence of a cell which expresses the corresponding antigen, thebacteriophage will bind to the cell. Bacteriophage which do not expressthe antibody will not bind to the cell. Such panning techniques are wellknown in the art and are described for example, in Wright et al.(supra).

Processes such as those described above, have been developed for theproduction of human antibodies using M13 bacteriophage display (Burtonet al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library isgenerated from mRNA obtained from a population of antibody-producingcells. The mRNA encodes rearranged immunoglobulin genes and thus, thecDNA encodes the same. Amplified cDNA is cloned into M13 expressionvectors creating a library of phage which express human Fab fragments ontheir surface. Phage which display the antibody of interest are selectedby antigen binding and are propagated in bacteria to produce solublehuman Fab immunoglobulin. Thus, in contrast to conventional monoclonalantibody synthesis, this procedure immortalizes DNA encoding humanimmunoglobulin rather than cells which express human immunoglobulin.

The procedures just presented describe the generation of phage whichencode the Fab portion of an antibody molecule. However, the inventionshould not be construed to be limited solely to the generation of phageencoding Fab antibodies. Rather, phage which encode single chainantibodies (scFv/phage antibody libraries) are also included in theinvention. Fab molecules comprise the entire Ig light chain, that is,they comprise both the variable and constant region of the light chain,but include only the variable region and first constant region domain(CH1) of the heavy chain. Single chain antibody molecules comprise asingle chain of protein comprising the Ig Fv fragment. An Ig Fv fragmentincludes only the variable regions of the heavy and light chains of theantibody, having no constant region contained therein. Phage librariescomprising scFv DNA may be generated following the procedures describedin Marks et al. (1991, J. Mol. Biol. 222:581-597). Panning of phage sogenerated for the isolation of a desired antibody is conducted in amanner similar to that described for phage libraries comprising Fab DNA.

The invention should also be construed to include synthetic phagedisplay libraries in which the heavy and light chain variable regionsmay be synthesized such that they include nearly all possiblespecificities (Barbas, 1995, Nature Medicine 1:837-839; de Kruif et al.1995, J. Mol. Biol. 248:97-105).

VII. Compositions

The invention includes a composition comprising an isolated nucleiccomplementary to a nucleic acid, or a portion thereof, encoding amammalian renalase, which is in an antisense orientation with respect totranscription. Preferably, the composition comprises a pharmaceuticallyacceptable carrier.

The invention includes a composition comprising an isolated mammalianrenalase polypeptide as described herein. Preferably, the compositioncomprises a pharmaceutically-acceptable carrier.

The invention also includes a composition comprising an antibody thatspecifically binds renalase. Preferably, the composition comprises apharmaceutically-acceptable carrier.

The invention further includes a composition comprising an isolatednucleic acid encoding a mammalian renalase. Preferably, the compositioncomprises a pharmaceutically acceptable carrier.

The compositions can be used to administer renalase to a cell, a tissue,or an animal or to inhibit expression of renalase in a cell, a tissue,or an animal. The compositions are useful to treat a disease, disorderor condition mediated by altered expression of renalase such thatdecreasing or increasing renalase expression or the level of the proteinin a cell, tissue, or animal, is beneficial to the animal. That is,where a disease, disorder or condition in an animal is mediated by orassociate with altered level of renalase expression or protein level,the composition can be used to modulate such expression or protein levelof renalase.

For administration to the mammal, a polypeptide, or a nucleic acidencoding it, and/or an antisense nucleic acid complementary to all or aportion thereof, can be suspended in any pharmaceutically acceptablecarrier, for example, HEPES buffered saline at a pH of about 7.8.

Other pharmaceutically acceptable carriers which are useful include, butare not limited to, glycerol, water, saline, ethanol and otherpharmaceutically acceptable salt solutions such as phosphates and saltsof organic acids. Examples of these and other pharmaceuticallyacceptable carriers known to those skilled in the art are described inRemington's Pharmaceutical Sciences (1991, Mack Publication Co., NewJersey).

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered, prepared, packaged, and/or sold informulations suitable for oral, rectal, vaginal, parenteral, topical,pulmonary, intranasal, buccal, ophthalmic, or another route ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations.

As used herein, “nanoparticle” is defined as a particle having adiameter of from 1 to 1000 nanometers, having any size, shape ormorphology. The nanoparticle may even be a “nanoshell,” which is ananoparticle having a discrete dielectric or semiconducting core sectionsurrounded by one or more conducting shell layers. A “nanoshell” is asubspecies of nanoparticles characterized by the discrete core/shellstructure. Both nanoshells and nanoparticles may contain dopants forbinding to, e.g., negatively charged molecules such as DNA, RNA and thelike. Examples of commonly used, positively charged dopands include Pr³,Er³, and Nd³. As used herein, “shell” means one or more shells that willgenerally surround at least a portion of one core. Several cores may beincorporated into a larger nanoshell. In one embodiment, thenanoparticles are administered to the animal using standard methods.

As used herein, the term “delivering” nanoparticles is used to describethe placement of the nanoparticles attached to, next to, or sufficientlyclose to the target location, e.g., intravenously, in order to maximizethe number of particles that will be able to contact cells at the targetlocation.

The compositions of the present invention include nucleic acid sequencesbound in, to or about nanoparticles, and methods for their use. Thebound nanoparticles can be used for the delivery of the nucleic acidsequences to a variety of biological targets, such as endothelial cells.In one embodiment, the nucleic acids, e.g., a nucleic acid gene underthe control of a promoter for gene expression is attached to apositively doped nanocore, which is then surrounded by a shell thatincludes a targeting ligand that is specific for a ligand target on,e.g., a cell of interest.

One embodiment of the invention relates to nucleic acid sequences boundin/to a nanoparticle. The nanoparticle is prepared by assembly of a“nanoparticle precursor.” The nucleic acid sequence can generally be anynucleic acid sequence selected for delivery into a biological target.The nucleic acid sequence can be DNA, RNA, PNA, or other synthetic ormodified nucleic acid sequences. In one embodiment, the nucleic acidsequence is a DNA sequence encoding human renalase (GenBank AccessionNo. BC 005364). The DNA sequence may be a naturally occurring sequence,a modified version of a naturally occurring sequence, or a syntheticsequence. In one embodiment, the nucleic acid is modified to maximizethe percentage of codon usage of the target host. The naturallyoccurring sequence may be a human, monkey, cow, pig, horse, cat, dog,rat, mouse, bear, rabbit, moose, fish, sheep, or other animal sequence.The sequence may be modified to add or delete particular sequences. Forexample, a DNA sequence could be modified to, e.g., remove restrictionsites, eliminate common cleavage or mutation sites, maximize binding toa nanocore. The sequence may be further modified to include additionalsequences that aid in transcription, translation, localization,elimination of protein cleavage sites, addition of cleavage and/orprocessing sites, and addition or removal of glycosylation sites.

In one embodiment, the nanoparticle precursor includes a nucleic acidsequence bound to a nanoparticle polymer. The bond between ananoparticle precursor and a nucleic acid may be non-covalent orcovalent. The nanoparticle polymer may be any polymer that can assembleinto a nanoparticle. For example, the nucleic acid sequence can benon-covalently bound to a first polymer. This first polymer can be a DNAbinding cationic polymer such as polyethyleneimine (“PEI”). The firstpolymer can be covalently bound to a second polymer. The second polymercan be a hydrophilic polymer such as polyethylene glycol (PEG). Forexample, the second polymer can be conjugated to a fraction of theprimary amines of PEI.

The hydrophilic polymer can be bound to a ligand such as an antibody.The antibody can be a polyclonal antibody or a monoclonal antibody, witha monoclonal antibody being presently preferred. The antibody can bespecific for a biological receptor or other cellularly expressedprotein. For example, the antibody can bind the lectin-like oxidized lowdensity lipoprotein (LDL) receptor-1, Lox-1. Antibodies provideattractive binding abilities, but have relatively high steric bulk.Smaller antibody fragments or other binding peptides or molecules may beused as a ligand in various embodiments of the invention.

When the nanoparticle precursor self-assembles, the nucleic acidmolecule is encapsulated within the formed nanoparticle, and theantibody or ligand is presented on the surface of the nanoparticle. Theencapsulated nucleic acid molecule is partially or fully protected fromdegradation by the environment, enzymes, hydrolysis, or other degradingforces.

The assembled nanoparticle can generally have an average diameter ofabout 1 nm to about 1000 nm. More narrow ranges of diameters includeabout 10 nm to about 250 nm, and about 40 nm to about 100 nm.

An additional embodiment of the invention relates to the assemblednanoparticle. The assembled nanoparticle comprises nanoparticleprecursors that have assembled in solution. The assembled nanoparticlespreferably contain nucleic acid sequences in the internal volume of thenanoparticles, and antibodies or other binding peptides presented on theexternal face of the nanoparticles. The nanoparticles can generally beany shape, with about spherical being presently preferred. Theantibodies preferably maintain their natural conformation, allowingbinding to their natural targets.

The assembled nanoparticles can be present in a variety of formulationsincluding in solution, dried, in liposomes, and so on. Specific examplesof formulations include fullerene nanoparticles, aqueous nanoparticlescomprised of oppositely charged polymers polyethylenimine (PEI) anddextran sulfate (DS) with zinc as a stabilizer, calcium phosphatenanoparticles, end-capped oligomers derived fromTris(hydroxymethyl)aminomethane bearing either a hydro- or afluorocarbon tail; conjugated poly(aminopoly(ethyleneglycol)cyanoacrylate-co-hexadecyl cyanoacrylate(poly(H(2)NPEGCA-co-HDCA) nanoparticles, biodegradable nanoparticlesformulated from poly (D,L-lactide-co-glycolide) (PLGA), and watersoluble, biodegradable polyphosphoester, poly(2-aminoethyl propylenephosphate) (PPE-EA) nanoparticles.

Aspects of the invention also relate to methods of preparing theassembled nanoparticles. The methods can comprise formation of a polymerconjugate, and contacting the polymer conjugate with nucleic acid toform a nanoparticle. The polymer conjugate includes a first polymer, asecond polymer, and a ligand. The first polymer preferably binds in anon-covalent manner to nucleic acids. A presently preferred firstpolymer is a DNA binding cationic polymer such as polyethyleneimine(“PEI”). The second polymer can be a hydrophilic polymer such aspolyethylene glycol (“PEG”). The ligand is presently preferred to be anantibody.

It is presently preferred that the parts of the polymer conjugate beconnected by covalent bonds. The specific order of assembly of thepolymer conjugate can be varied. For example, the first polymer andsecond polymer can be connected, then the ligand can be connected.Alternatively, the second polymer and the ligand can be connected, thenthe first polymer can be connected. The methods can further comprise anisolation or purification step to be performed after the contactingstep. The described assembled nanoparticles can be used in a variety ofapplications. The nanoparticles can be used in in vitro or in vivoapplications.

The present invention also provides a pharmaceutical composition ofrenalase in a microcrystalline form. Various methods for obtainingprotein crystals have been developed, including the free interfacediffusion method (Salemme, F. R. (1972) Arch. Biochem. Biophys.151:533-539), vapor diffusion in the hanging or sitting drop method(McPherson, A. (1982) Preparation and Analysis of Protein Crystals, JohnWiley and Son, New York, pp 82-127), and liquid dialysis (Bailey, K.(1940) Nature 145:934-935). Compared to non-crystalline proteins,crystalline proteins provide significant improvements in stability andconcentration of proteins which leads to the opportunity for oral andparenteral delivery of proteins. In some instances, particularcrystalline forms of a molecule may have more bioactive, dissolvefaster, decompose less readily, and/or be easier to purify.

Proteins, glycoproteins, enzymes, antibodies, hormones and peptidecrystals or crystal formulations can be encapsulated into compositionsfor biological delivery to humans and animals. Methods for crystallizingproteins, preparing stabilized formulations using pharmaceuticalingredients or excipients and optionally encapsulating them in apolymeric carrier to produce compositions and using such protein crystalformulations and compositions for biomedical applications, includingdelivery of therapeutic proteins and vaccines are well known in the art.For example, U.S. Pat. No. 6,541,606, discloses that protein crystals orcrystal formulations are encapsulated within a matrix comprising apolymeric carrier to form a composition. The formulations andcompositions enhance preservation of the native biologically activetertiary structure of the proteins and create a reservoir which canslowly release active protein where and when it is needed. Suchpolymeric carriers include biocompatible and biodegradable polymers. Thebiologically active protein is subsequently released in a controlledmanner over a period of time, as determined by the particularencapsulation technique, polymer formulation, crystal geometry, crystalsolubility, crystal crosslinking and formulation conditions used.

The compositions of the invention may be administered via numerousroutes, including, but not limited to, oral, rectal, vaginal,parenteral, topical, pulmonary, intranasal, buccal, or ophthalmicadministration routes. The route(s) of administration will be readilyapparent to the skilled artisan and will depend upon any number offactors including the type and severity of the disease being treated,the type and age of the veterinary or human patient being treated, andthe like.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in oral solid formulations,ophthalmic, suppository, aerosol, topical or other similar formulations.In addition to the compound such as heparan sulfate, or a biologicalequivalent thereof, such pharmaceutical compositions may containpharmaceutically-acceptable carriers and other ingredients known toenhance and facilitate drug administration. Other possible formulations,such as nanoparticles, liposomes, resealed erythrocytes, andimmunologically based systems may also be used to administer renalaseand/or a nucleic acid encoding the same according to the methods of theinvention.

Compounds which are identified using any of the methods described hereinmay be formulated and administered to a mammal for treatment of arterialrestenosis, adventitial fibrosis, fibrosis in any organ or tissue,negative remodeling, excessive bone formation, excessive ossification,and the like, are now described. The invention encompasses thepreparation and use of pharmaceutical compositions comprising a compounduseful for treatment of arterial restenosis, adventitial fibrosis,negative remodeling, and the like, as an active ingredient. Such apharmaceutical composition may consist of the active ingredient alone,in a form suitable for administration to a subject, or thepharmaceutical composition may comprise the active ingredient and one ormore pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject. As used herein, the term“physiologically acceptable” ester or salt means an ester or salt formof the active ingredient which is compatible with any other ingredientsof the pharmaceutical composition, which is not deleterious to thesubject to which the composition is to be administered.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, ophthalmic, intrathecal or another route of administration.Other contemplated formulations include projected nanoparticles,liposomal preparations, resealed erythrocytes containing the activeingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyamide and cyanatescavengers.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water. A tablet comprising the active ingredient may, forexample, be made by compressing or molding the active ingredient,optionally with one or more additional ingredients. Compressed tabletsmay be prepared by compressing, in a suitable device, the activeingredient in a free-flowing form such as a powder or granularpreparation, optionally mixed with one or more of a binder, a lubricant,an excipient, a surface active agent, and a dispersing agent. Moldedtablets may be made by molding, in a suitable device, a mixture of theactive ingredient, a pharmaceutically acceptable carrier, and at leastsufficient liquid to moisten the mixture. Pharmaceutically acceptableexcipients used in the manufacture of tablets include, but are notlimited to, inert diluents, granulating and disintegrating agents,binding agents, and lubricating agents. Known dispersing agents include,but are not limited to, potato starch and sodium starch glycollate.Known surface active agents include, but are not limited to, sodiumlauryl sulphate. Known diluents include, but are not limited to, calciumcarbonate, sodium carbonate, lactose, microcrystalline cellulose,calcium phosphate, calcium hydrogen phosphate, and sodium phosphate.Known granulating and disintegrating agents include, but are not limitedto, corn starch and alginic acid. Known binding agents include, but arenot limited to, gelatin, acacia, pre-gelatinized maize starch,polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Knownlubricating agents include, but are not limited to, magnesium stearate,stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin. Soft gelatincapsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.,polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e., about 20° C.) and which isliquid at the rectal temperature of the subject (i.e., about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition may be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or gel or cream or a solution for vaginalirrigation.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made bycombining the active ingredient with a pharmaceutically acceptableliquid carrier. As is well known in the art, douche preparations may beadministered using, and may be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject. Douche preparations mayfurther comprise various additional ingredients including, but notlimited to, antioxidants, antibiotics, antifungal agents, andpreservatives.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen-free water) prior toparenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken, i.e., by rapid inhalation throughthe nasal passage from a container of the powder held close to thenares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 nanometers, and may further comprise one or moreof the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other ophthalmalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifingalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed. (1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa.), which isincorporated herein by reference.

Typically, dosages of the compound of the invention which may beadministered to an animal, preferably a human, will vary depending uponany number of factors, including but not limited to, the type of animaland type of disease state being treated, the age of the animal and theroute of administration.

The compound can be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even leesfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, etc. Vectors for Use in Practicing the Invention

VIII. Methods

A. Methods of Treating or Alleviating a Disease, Disorder or ConditionAssociated with or Mediated by Renalase Expression

In one aspect of the present invention, there is provided a method totreat diseases, disorders, and conditions associated with or mediated bymammalian renalase. Such disease, disorders and conditions include, butare not limited to, ESRD, chronic hypertension, systolic hypertension,isolated systolic hypertension, diabetic hypertension, pulmonaryhypertension, acute severe hypertension, asymptomatic left ventriculardysfunction, chronic congestive heart failure (CHF), myocardialinfarction (MI), cardiac rhythm disturbance, stroke, atherosclerosis,depression, anxiety, mania and schizophrenia and the like.

The data disclosed herein suggest that over-expression of renalase,under-expression of renalase, overactive renalase, and inactive renalaseare associated with various diseases, disorders or conditions such thatmethods of decreasing the level of renalase, increasing the level ofrenalase, decreasing the activity of renalase, and increasing theactivity of renalase can potentially produce benefits, wherein thechosen method depends on the specific disorder, condition and/or diseaseunder consideration. Therefore, a method of affecting the level ofrenalase to treat/alleviate a wide plethora of diseases, disorders, orconditions is disclosed herein, where the level of renalase is eitherdecreased or increased compared with the level of renalase in the cellprior to treatment, or compared with the level of renalase in anotherwise identical cell that is obtained from a mammal known not to beafflicted with a disease, disorder or condition associated with, ormediated by, an altered level of renalase.

Whether expression of renalase, levels of the polypeptide, or itsactivity, is increased or decreased, one skilled in the art wouldappreciate, based on this disclosure, that methods of reducing orinducing renalase encompass administering naturally occurring ornon-naturally occurring renalase, a renalase inhibitor, or a recombinantcell that either expresses or lacks expression of renalase. Thus, oneskilled in the art would appreciate, based on the disclosure providedherein, that the present invention encompasses methods of treatmentsknown in the art to effect either a detectable increase or decrease inthe level of renalase expression or activity in a mammal.

The compositions of the present invention can be used to administerrenalase to a cell, a tissue, or an animal or to promote expression ofrenalase in a cell, a tissue, or an animal. The compositions are usefulto treat a disease, disorder or condition mediated by altered expressionof renalase such that decreasing or increasing renalase expression orthe level of the protein in a cell, tissue, or animal, is beneficial tothe animal. That is, where a disease, disorder or condition in an animalis mediated by or associate with altered level of renalase expression orprotein level, the composition can be used to modulate such expressionor protein level of renalase.

One skilled in the art would understand, based upon the disclosureprovided herein, that it may be useful to increase the level or activityof renalase in a cell. That is, the data disclosed herein demonstratingthe association between renalase expression and hypertension, indicatethat overexpression or an increase in renalase activity can lower bloodpressure. This can be useful to treat or alleviate a disease, disorderof condition associated with or mediated by decreased expression, level,or activity of renalase (e.g., hypertension), when compared to theexpression, level or activity of renalase in otherwise identical cell,tissue, or animal that does not suffer from the disease, disorder orcondition, by administering naturally occurring or non-naturallyoccurring renalase. Such diseases, disorders or conditions include, butare not limited to ESRD, hypertension, a cardiovascular disorder, or amental disorder.

The data disclosed herein demonstrate that renalase can regulatesystemic blood pressure. Thus, the skilled artisan would appreciate,based upon the disclosure provided herein, that renalase, by metabolizecatecholamines, can be used to treat cardiovascular diseases. Therefore,the present invention encompasses a method of increasing catecholaminemetabolism comprising increasing the activity of renalase in circulationor the expression of renalase in a cell. For example, ESRD can betreated by administering to a mammal an effective amount of renalase.This is because, the data disclosed herein demonstrate that renalase isassociated with ESRD. That is, the data demonstrate that lack ofexpression of renalase is correlated with ESRD. Further, the datadisclosed herein demonstrate that infusion of recombinant renalase intorats results in a reduction in the rats' blood pressure, suggesting thatrenalase can be used to treat ESRD induced hypertension and othercardiovascular diseases.

In yet another embodiment, the invention includes a method ofalleviating a disease, disorder or condition mediated by alteredexpression or activity of renalase by administering to a mammal aninhibitor that inhibits renalase expression and/or activity. Just likeMAO inhibitors, a renalase inhibitor may prove useful in the treatmentof CNS disorders such as mood disorders. Therefore, decreasingexpression of renalase or decreasing activity of renalase with, forexample, a chemical compound, a peptidomimetic, a small molecule,ribozymes, antisense nucleic acids, antibodies, and intrabodies thatinhibit renalase, will increase monoamine content in the brain leadingto the alleviation of the symptoms of these disorders. Thus, one ofordinary skill in the art would understand that inhibiting renalase,which can be accomplished by a variety of methods as more fully setforth elsewhere herein, is a useful treatment for CNS disorders, inparticular mood disorders.

The CNS disorders include, but art not limited to, dementia, Alzheimer'sdisease, schizophrenia, psychosis, depression, headaches, and migraineheadache. As used herein, the term “depression” includes depressivedisorders, for example, single episodic or recurrent major depressivedisorders, and dysthymic disorders, depressive neurosis, and neuroticdepression; melancholic depression including anorexia, weight loss,insomnia and early morning waking, and psychomotor retardation; atypicaldepression (or reactive depression) including increased appetite,hypersomnia, psychomotor agitation or irritability, anxiety and phobias;seasonal affective disorder; or bipolar disorders or manic depression,for example, bipolar I disorder, bipolar II disorder and cyclothymicdisorder.

Other mood disorders encompassed within the term “depression” includedysthymic disorder with early or late onset and with or without atypicalfeatures; dementia of the Alzheimer's type, with early or late onset,with depressed mood; vascular dementia with depressed mood; mooddisorders induced by alcohol, amphetamines, cocaine, hallucinogens,inhalants, opioids, phencyclidine, sedatives, hypnotics, anxiolytics andother substances; schizoaffective disorder of the depressed type; andadjustment disorder with depressed mood.

The compositions of the present invention are useful for the treatmentof anxiety. As used herein, the term “anxiety” includes anxietydisorders, such as panic disorder with or without agoraphobia,agoraphobia without history of panic disorder, specific phobias, forexample, specific animal phobias, social phobias, obsessive-compulsivedisorder, stress disorders including post-traumatic stress disorder andacute stress disorder, and generalized anxiety disorders.

“Generalized anxiety” is typically defined as an extended period (e.g.at least six months) of excessive anxiety or worry with symptoms on mostdays of that period. The anxiety and worry is difficult to control andmay be accompanied by restlessness, being easily fatigued, difficultyconcentrating, irritability, muscle tension, and disturbed sleep.

“Panic disorder” is defined as the presence of recurrent panic attacksfollowed by at least one month of persistent concern about havinganother panic attack. A “panic attack” is a discrete period in whichthere is a sudden onset of intense apprehension, fearfulness or terror.During a panic attack, the individual may experience a variety ofsymptoms including palpitations, sweating, trembling, shortness ofbreath, chest pain, nausea and dizziness. Panic disorder may occur withor without agoraphobia.

“Phobias” includes agoraphobia, specific phobias and social phobias.“Agoraphobia” is characterized by an anxiety about being in places orsituations from which escape might be difficult or embarrassing or inwhich help may not be available in the event of a panic attack.Agoraphobia may occur without history of a panic attack. A “specificphobia” is characterized by clinically significant anxiety provoked byexposure to a specific feared object or situation. Specific phobiasinclude the following subtypes: animal type, cued by animals or insects;natural environment type, cued by objects in the natural environment,for example storms, heights or water; blood-injection-injury type, cuedby the sight of blood or an injury or by seeing or receiving aninjection or other invasive medical procedure; situational type, cued bya specific situation such as public transportation, tunnels, bridges,elevators, flying, driving or enclosed spaces; and other type where fearis cued by other stimuli. Specific phobias may also be referred to assimple phobias. A “social phobia” is characterized by clinicallysignificant anxiety provoked by exposure to certain types of social orperformance circumstances. Social phobia may also be referred to associal anxiety disorder.

Other anxiety disorders encompassed within the term “anxiety” includeanxiety disorders induced by alcohol, amphetamines, caffeine, cannabis,cocaine, hallucinogens, inhalants, phencyclidine, sedatives, hypnotics,anxiolytics and other substances, and adjustment disorders with anxietyor with mixed anxiety and depression.

Anxiety may be present with or without other disorders such asdepression in mixed anxiety and depressive disorders. The compositionsof the present invention are therefore useful in the treatment ofanxiety with or without accompanying depression.

The invention further provides a method of alleviating a disease,disorder or condition mediated by altered expression of renalase byadministering an antisense nucleic acid complementary to a nucleic acidencoding renalase to a patient afflicted with a disease, disorder orcondition mediated by increased renalase expression compared to thelevel of renalase expression in otherwise identical but normal tissue,i.e., tissue which does not exhibit any detectable clinical parametersassociated with the disease, disorder or condition being treated oralleviated. This, in turn, mediates a decrease in renalase expressionthereby alleviating a disease, disorder or condition mediated byabnormal expression of renalase.

Although inhibition of renalase is exemplified by administering anantisense to a cell thereby inhibiting expression of renalase in thecell, one skilled in the art would appreciate that there are a wideplethora of methods for inhibiting protein expression and/or activity ina cell. Such methods include, but are not limited to, inhibitingexpression of renalase using ribozymes, and inhibiting activity of theprotein in a cell by, for instance, administering an antibody to thecell by, e.g., administering a nucleic acid encoding the antibody to thecell such that the antibody is expressed in the cell thus delivering theantibody to the cell cytosol. The use of these “intrabodies” to inhibitthe intracellular activity of a protein are well-known in the art. See,e.g., Verma et al. (1997, Nature 389:239-242; Benhar & Pastan, 1995, J.Biol. Chem. 270:23373-23380; Willuda et al., 1999, Cancer Res.59:5758-5767; and Worn et al., 2000, J. Biol. Chem. 275:2795-2803).Therefore, the present invention encompasses any method of inhibitingthe activity of a protein of interest in a cell using such methods asare known in the art or to be developed in the future.

In another embodiment of the invention, an individual suffering from adisease, disorder or a condition that is associated with or mediated byrenalase expression can be treated by supplementing, augmenting and/orreplacing defective cells with cells that lack renalase expression. Thecells can be derived from cells obtained from a normal syngeneic matcheddonor or cells obtained from the individual to be treated. The cells maybe genetically modified to inhibit renalase expression. Alternatively,the cells can be modified to increase renalase expression usingrecombinant methods well-known in the art. Also, the inventionencompasses using normal cells obtained from an otherwise identicaldonor that does not suffer from any disease or disorder associated withaltered renalase expression, which cells can be administered to a mammalin need thereof.

Additionally, the invention includes ex vivo techniques where a cell isobtained from the mammal, modified to express increased or decreasedlevel of renalase, and reintroduced into the mammal. Moreover, cellsfrom the mammal which express a normal level of renalase, compared withthe level of renalase expressed in an otherwise identical cell obtainedfrom a like mammal not suffering from any condition associated withaltered renalase expression, can be grown and expanded and an effectivenumber of the cells can be reintroduced into the mammal. Such methodsinclude cell and gene therapy techniques relating to use of bone marrowstromal cells which methods are well-known in the art. Thus, one skilledin the art would appreciate that cell therapy and gene therapy relatingto cells that have or lack detectable renalase expression wherein thecells are administered in vivo are encompassed in the present invention.

In addition to replacing defective cells with repaired cells or normalcells from syngeneic, immunologically-matched donors, the method of theinvention may also be used to facilitate expression of a desired proteinthat when secreted in an animal, has a beneficial effect. That is, cellsmay be isolated, furnished with a gene encoding renalase and introducedinto the donor or into a syngeneic matched recipient wherein expressionof exogenous renalase exerts a therapeutic effect.

One skilled in the art would understand, based upon the disclosureprovided herein, that secretion of renalase from a cell is contemplatedin the present invention. That is, the routineer would appreciate, basedupon the disclosure provided herein, that secretion of renalase from acell can be a useful therapeutic method and that the present inventionincludes secretion of renalase from a cell. Secretion of renalase from acell can be effected according to standard methods well-known in the artand methods to be developed in the future. Such methods include, but arenot limited to, covalently linking a nucleic acid encoding a signalpeptide of a secreted molecule to the 5′ end of an isolated nucleic acidencoding renalase. A wide plethora of signal sequences that can be usedto mediate secretion of a protein from a cell are available andwell-known in the art and the invention includes those as well assequences to be developed in the future to drive secretion of a proteinfrom a cell.

This aspect of the invention relates to gene therapy in whichtherapeutic amounts of renalase are administered to an individual. Thatis, according to some aspects of the present invention, recombinantcells transfected with either nucleic acid encoding renalase, antisensenucleic acids, or a knock-out targeting vector of the invention, can beused as cell therapeutics to treat a disease, disorder or a conditioncharacterized by altered expression of renalase, including the lack ofexpression of renalase.

In particular, a gene construct that comprises a heterologous gene whichencodes renalase is introduced into cells. These recombinant cells areused to purify isolated renalase, which was is administered to ananimal. One skilled in the art would understand, based upon thedisclosure provided herein, that instead of administering an isolatedrenalase polypeptide, renalase can be administered to a mammal in needthereof by administering to the mammal the recombinant cells themselves.This will benefit the recipient individual who will benefit when theprotein is expressed and secreted by the recombinant cell into therecipient's system.

According to the present invention, gene constructs comprisingnucleotide sequences of the invention are introduced into cells. Thatis, the cells, referred to herein as “recombinant cells,” aregenetically altered to introduce a nucleic acid encoding renalase or anucleic acid that inhibits renalase expression in and/or secretion bythe recombinant cell (e.g., an antisense renalase nucleic acid, anucleic acid encoding an anti-renalase antibody, and the like), therebymediating a beneficial effect on an recipient to which the recombinantcell is administered. According to some aspects of the invention, cellsobtained from the same individual to be treated or from anotherindividual, or from a non-human animal, can be genetically altered toreplace a defective renalase gene and/or to introduce a renalase genewhose expression has a beneficial effect on the individual, or toinhibit renalase expression which can have a beneficial effect on theindividual.

In some aspects of the invention, an individual suffering from adisease, disorder or a condition can be treated by supplementing,augmenting and/or replacing defective or deficient nucleic acid encodingrenalase by providing an isolated recombinant cells containing geneconstructs that include normal, functioning copies of a nucleic acidencoding renalase. This aspect of the invention relates to gene therapyin which the individual is provided with a nucleic encoding renalase forwhich they are deficient in presence and/or function. The isolatednucleic acid encoding renalase provided by the cell compensates for thedefective renalase expression of the individual, because, when thenucleic acid is expressed in the individual, a protein is produced whichserves to alleviate or otherwise treat the disease, disorder orcondition in the individual. Such nucleic acid preferably encodes arenalase polypeptide that is secreted from the recombinant cell.

In all cases in which a gene construct encoding renalase is transfectedinto a cell, the nucleic acid is operably linked to an appropriatepromoter/regulatory sequence which is required to achieve expression ofthe nucleic acid in the recombinant cell. Such promoter/regulatorysequences include but are not limited to, constitutive and inducibleand/or tissue specific and differentiation specific promoters, and arediscussed elsewhere herein. Constitutive promoters include, but are notlimited to, the cytomegalovirus immediate early promoter and the Roussarcoma virus promoter. In addition, housekeeping promoters such asthose which regulate expression of housekeeping genes may also be used.Other promoters include those which are preferentially expressed incells of the central nervous system, such as, but not limited thepromoter for the gene encoding glial fibrillary acidic protein. Inaddition, promoter/regulatory elements may be selected such that geneexpression is inducible. For example, a tetracycline inducible promotermay be used (Freundlich et al., 1997, Meth. Enzymol. 283:159-173).

The gene construct is preferably provided as an expression vector whichincludes the coding sequence of a mammalian renalase of the inventionoperably linked to essential promoter/regulatory sequences such thatwhen the vector is transfected into the cell, the coding sequence isexpressed by the cell. The coding sequence is operably linked to thepromoter/regulatory elements necessary for expression of the sequence inthe cells. The nucleotide sequence that encodes the protein may be cDNA,genomic DNA, synthesized DNA or a hybrid thereof or an RNA molecule suchas mRNA. The gene construct, which includes the nucleotide sequenceencoding renalase operably linked to the promoter/regulatory elements,may remain present in the cell as a functioning episomal molecule or itmay integrate into the chromosomal DNA of the cell. Genetic material maybe introduced into cells where it remains as separate genetic materialin the form of a plasmid. Alternatively, linear DNA which can integrateinto a host cell chromosome may be introduced into the cell. Whenintroducing DNA into the cell, reagents which promote DNA integrationinto chromosomes may be added. DNA sequences which are useful to promoteintegration may also be included in the DNA molecule. Alternatively, RNAmay be introduced into the cell.

In order for genetic material in an expression vector to be expressed,the promoter/regulatory elements must be operably linked to thenucleotide sequence that encodes the protein. In order to maximizeprotein production, promoter/regulatory sequences may be selected whichare well suited for gene expression in the desired cells. Moreover,codons may be selected which are most efficiently transcribed in thecell. One having ordinary skill in the art can produce recombinantgenetic material as expression vectors which are functional in thedesired cells.

It is also contemplated that promoter/regulatory elements may beselected to facilitate tissue specific expression of the protein. Thus,for example, specific promoter/regulatory sequences may be provided suchthat the heterologous gene will only be expressed in the tissue wherethe recombinant cells are implanted. Additionally, the skilled artisanwould appreciate, based upon the disclosure provided herein, that therenalase promoter can be operably linked to a nucleic acid of interestthereby directing the expression of the nucleic acid at the site oftissue or organ. Similarly, the renalase promoter can drive expressionof a nucleic acid of interest where such expression is beneficial wherehigh catecholamine circulation is a problem.

One skilled in the art would understand, based upon the disclosureprovided herein, that the preferred tissues where the expression or lackof expression of renalase is to be targeted include, but are not limitedto, ulcerations of the skin, bone fractures, and the like. In addition,promoter/regulatory elements may be selected such that gene expressionis inducible. For example, a tetracycline inducible promoter may be used(Freundlich et al., 1997, Meth. Enzymol. 283:159-173).

Without wishing to be bound by any particular theory, the nucleic acidencoding renalase preferably includes a putative signal sequence asdisclosed elsewhere herein (e.g., amino acids at N terminus of humanrenalase), which may direct the transport and secretion of the renalaseencoded by the isolated nucleic acid in the recombinant cell. The signalsequence is likely processed and removed upon secretion of the maturerenalase protein from the cell. Alternatively, without wishing to bebound by any particular theory, the putative signal sequence may not becleaved, but may instead be a transmembrane domain.

In addition to providing cells with recombinant genetic material thateither corrects a genetic defect in the cells, that encodes a proteinwhich is otherwise not present in sufficient quantities and/orfunctional condition so that the genetic material corrects a geneticdefect in the individual, and/or that encodes a protein which is usefulas beneficial in the treatment or prevention of a particular disease,disorder or condition associated therewith, and that inhibits expressionof renalase in the cell (e.g., a knock-out targeting vector, anantisense nucleic acid, and the like), genetic material can also beintroduced into the recombinant cells used in the present invention toprovide a means for selectively terminating such cells should suchtermination become desirable. Such means for targeting recombinant cellsfor destruction may be introduced into recombinant cells.

According to the invention, recombinant cells can be furnished withgenetic material which renders them specifically susceptible todestruction. For example, recombinant cells may be provided with a genethat encodes a receptor that can be specifically targeted with acytotoxic agent. An expressible form of a gene that can be used toinduce selective cell death can be introduced into the recombinantcells. In such a system, cells expressing the protein encoded by thegene are susceptible to targeted killing under specific conditions orin, the presence or absence of specific agents. For example, anexpressible form of a herpes virus thymidine kinase (herpes tk) gene canbe introduced into the recombinant cells and used to induce selectivecell death. When the introduced genetic material that includes theherpes tk gene is introduced into the individual, herpes tk will beproduced. If it is desirable or necessary to kill the implantedrecombinant cells, the drug gangcyclovir can be administered to theindividual which will cause the selective killing of any cell producingherpes tk. Thus, a system can be provided which allows for the selectivedestruction of implanted recombinant cells.

One skilled in the art would understand, based upon the disclosureprovided herein, that the present invention encompasses production ofrecombinant cells to either provide renalase to or inhibit renalaseexpression in a mammal. That is, the cells can be used to administerrenalase to an animal or to deliver a molecule (e.g., a knock-outtargeting vector, an antisense nucleic acid, a ribozyme, and antibodythat specifically binds with renalase, and the like).

Administration of renalase to an animal can be used as a model system tostudy the mechanism of action of renalase or to develop model systemsuseful for the development of diagnostics and/or therapeutics fordiseases, disorders or conditions associated with renalase expression.

Further, the delivery of renalase to an animal mediated byadministration of recombinant cells expressing and secreting renalasecan also be used to treat or alleviate a disease, disorder or conditionwhere increasing the level of renalase mediates a therapeutic effect.More specifically, administration of renalase to an animal byadministering a recombinant cell expressing a nucleic acid encodingrenalase can be useful for treatment of ESRD, hypertension,cardiovascular diseases, among other things.

Alternatively, administration of recombinant cells comprising a nucleicacid the expression of which inhibits or reduces renalase expression,activity, and/or secretion from a cell, can be used as a model for thedevelopment of diagnostics and/or therapeutics useful for diseases,disorders or conditions associated with or mediated by renalaseexpression, activity, and/or secretion. The present inventionencompasses that the recombinant cells can produce the molecule thatinhibits renalase expression thereby providing such molecule to theanimal. Alternatively, without wishing to be bound by any particulartheory, the recombinant cells themselves, which are otherwise functionalcells, except for the inability to express renalase, can perform thefunctions of otherwise identical but non-recombinant cells, withoutbeing subject to the renalase signaling pathway.

Cells, both obtained from an animal, from established cell lines thatare commercially available or to be developed, or primary cells culturedin vitro, can be transfected using well known techniques readilyavailable to those having ordinary skill in the art. Thus, the presentinvention is not limited to obtaining cells from a donor animal or fromthe patient animal itself. Rather, the invention includes using any cellthat can be engineered using a nucleic acid of the invention such thatthe recombinant cell either expresses renalase (where it did not expressrenalase prior to being engineered, or where the cell produced renalaseat a different level prior to the introduction of the nucleic acid intothe cell) or the recombinant cell does not express renalase or expressesit at a lower level (where it expressed renalase before or expressedrenalase at a different level prior to introduction of the nucleic acidinto the cell).

Nucleic acids can be introduced into the cells using standard methodswhich are employed for introducing a gene construct into cells whichexpress the protein encoded by the gene or which express a molecule thatinhibits renalase expression. In some embodiments, cells are transfectedby calcium phosphate precipitation transfection, DEAE dextrantransfection, electroporation, microinjection, liposome-mediatedtransfer, chemical-mediated transfer, ligand mediated transfer orrecombinant viral vector transfer.

In some embodiments, recombinant adenovirus vectors are used tointroduce DNA having a desired sequence into the cell. In someembodiments, recombinant retrovirus vectors are used to introduce DNAhaving a desired sequence into the cell. In some embodiments, standardcalcium phosphate, DEAE dextran or lipid carrier mediated transfectiontechniques are employed to incorporate a desired DNA into dividingcells. Standard antibiotic resistance selection techniques can be usedto identify and select transfected cells. In some embodiments, DNA isintroduced directly into cells by microinjection. Similarly, well knownelectroporation or particle bombardment techniques can be used tointroduce foreign DNA into cells. A second gene is usuallyco-transfected with and/or covalently linked to the nucleic acidencoding renalase, or knock-out targeting vector or antisense moleculethereto. The second gene is frequently a selectableantibiotic-resistance gene. Transfected recombinant cells can beselected by growing the cells in an antibiotic that kills cells that donot take up the selectable gene. In most cases where the two genes areunlinked and co-transfected, the cells that survive the antibiotictreatment contain and express both genes.

Methods for assessing the level of renalase (e.g., using anti-renalaseantibodies in Western blot or other immune-based analyses such as ELISA)and/or methods for assessing the level of renalase expression in a celland/or tissues (e.g., using Northern blot analysis, RT-PCR analysis, insitu hybridization, and the like) are disclosed herein or are well knownto those skilled in the art. Such assays can be used to determine the“effective amount” of renalase (whether using an isolated nucleic acid,antibody, antisense nucleic acid, ribozyme, recombinant cell, and thelike) to be administered to the animal in order to reduce or increasethe level of renalase to a desired level.

B. Methods of Identifying Useful Compounds

The present invention further includes a method of identifying acompound that affects expression and/or activity of renalase in a cell.The method comprises contacting a cell with a test compound andcomparing the level of expression and/or activity of renalase in thecell so contacted with the level of expression and/or activity ofrenalase in an otherwise identical cell not contacted with the compound.If the level of expression and/or activity of renalase is higher orlower in the cell contacted with the test compound compared to the levelof expression and/or activity of renalase in the otherwise identicalcell not contacted with the test compound, this is an indication thatthe test compound affects expression and/or activity of renalase in acell.

Similarly, the present invention includes a method of identifying acompound that reduces expression and/or activity of renalase in a cell.The method comprises contacting a cell with a test compound andcomparing the level of expression and/or activity of renalase in thecell contacted with the compound with the level of expression and/oractivity of renalase in an otherwise identical cell, which is notcontacted with the compound. If the level of expression and/or activityof renalase is lower in the cell contacted with the compound compared tothe level in the cell that was not contacted with the compound, thenthat is an indication that the test compound affects reduces expressionand/or activity of renalase in a cell.

One skilled in the art would appreciate, based on the disclosureprovided herein, that the level of expression and/or activity ofrenalase in the cell can be measured by determining the level ofexpression and/or activity of mRNA encoding renalase. Alternatively, thelevel of expression and/or activity of mRNA encoding renalase can bedetermined by using immunological methods to assess renalase productionfrom such mRNA as exemplified herein using Western blot analysis usingan anti-renalase antibody of the invention. Further, nucleic acid-baseddetection methods, such as Northern blot and PCR assays and the like,can be used as well. In addition, the level of renalase activity in acell can also be assessed by determining the level of various parameterswhich can be affected by renalase activity such as, for example, thelevel of renalase expression and/or activity in kidney, heart, skeletalmuscle, and small intestine, and the like. Alternatively, the level ofrenalase activity can be assessed in an amine oxidase assay. Thus, oneskilled in the art would appreciate, based upon the extensive disclosureand reduction to practice provided herein, that there are a plethora ofmethods which can be used to assess the level of expression and/oractivity of renalase in a cell including those methods disclosed herein,methods well-known in the art, and other methods to be developed in thefuture.

Further, one skilled in the art would appreciate based on the disclosureprovided herein that, as disclosed in the examples below, a cell whichlacks endogenous renalase expression and/or activity can be transfectedwith a vector comprising an isolated nucleic acid encoding renalasewhereby expression and/or activity of renalase is effected in the cell.The transfected cell is then contacted with the test compound therebyallowing the determination of whether the compound affects theexpression and/or activity of renalase. Therefore, one skilled in theart armed with the present invention would be able to, by selectivelytransfecting a cell lacking detectable levels of renalase usingrenalase-expressing vectors, identify a compound which selectivelyaffects renalase expression and/or activity.

The skilled artisan would further appreciate, based upon the disclosureprovided herein, that where an isolated nucleic acid encoding renalaseis administered to a cell lacking endogenous detectable levels ofrenalase expression and/or activity such that detectable renalase isproduced by the cell, the isolated nucleic acid can comprise anadditional nucleic acid encoding, e.g., a tag polypeptide, covalentlylinked thereto. This allows the detection of renalase expression and/oractivity by detecting the expression and/or activity of the tagpolypeptide. Thus, the present invention encompasses methods ofdetecting renalase expression and/or activity by detecting expressionand/or activity of another molecule which is co-expressed with renalase.

The invention includes a method of identifying a protein thatspecifically binds with renalase. Renalase binds with at least one otherprotein, whereby such interaction with other protein(s) may affect thebiological function of renalase. Thus, the invention encompassesmethods, which are well-known in the art or to be developed, foridentifying a protein that specifically binds with and/or associateswith renalase. Such methods include, but are not limited to, proteinbinding assays wherein the target protein, i.e., renalase, isimmobilized on an appropriate support and incubated under conditionsthat allow renalase binding with a renalase-associated protein. Renalasecan be immobilized on a support using standard methods such as, but notlimited to, production of renalase comprising aglutathione-S-transferase (GST) tag, a maltose binding protein (MBP)tag, or a His₆-tag, where the fusion protein is then bound toglutathione-Sepharose beads, a maltose-column, or a nickel chelationresin (e.g., His-Bind resin, Novagen, Madison, Wis.), respectively. Thesolid support is washed to remove proteins which may be non-specificallybound thereto and any renalase-associated protein can then bedissociated from the matrix thereby identifying a renalase-associatedprotein.

In addition, a protein that specifically binds with renalase, e.g., areceptor, a ligand, and/or other renalase-associated protein, can beidentified using, for example, a yeast two hybrid assay. Yeast twohybrid assay methods are well-known in the art and can be performedusing commercially available kits (e.g., MATCHMAKER™ Systems, ClontechLaboratories, Inc., Palo Alto, Calif., and other such kits) according tostandard methods. Therefore, once armed with the teachings providedherein, e.g., the full amino and nucleic acid sequences of the “bait”protein, renalase, one skilled in the art can easily identify a proteinthat specifically binds with renalase such as, but not limited to, arenalase receptor protein, a renalase ligand, and the like.

One skilled in the art would understand, based upon the disclosureprovided herein, that the invention encompasses any molecule identifiedusing the methods discussed elsewhere herein. That is, molecules thatassociate with renalase, such as but not limited to, a renalase receptorprotein, a renalase ligand protein, or both, can be used to developtherapeutics and diagnostics for diseases, disorders or conditionsmediated by renalase interaction with a renalase-associated protein suchas ESRD, hypertension, cardiovascular diseases, and the like. That is,one skilled in the art would appreciate, as more fully set forthelsewhere herein in discussing antibodies that specifically bind withrenalase, that a renalase-associated protein can be used to developtherapeutics that inhibit renalase activity in a cell by inhibitingnecessary renalase receptor/ligand interactions and other renalasebinding interactions, which are required for renalase activity.

Renalase-associated proteins identified by the above-disclosed methodscan be used directly to inhibit renalase interactions by contacting acell with the renalase-associated protein, or a portion thereof, or theycan be used to develop antibodies and/or peptidomimetics that caninhibit the renalase-associated interaction with renalase therebyinhibiting renalase function and activity. Thus, renalase-associatedproteins, including a renalase receptor/ligand protein, are useful andare encompassed by the invention.

C. Methods of Diagnosis and Assessment of Therapies

The present invention includes methods of diagnosis certain diseases,disorders, or conditions such as, but not limited to ESRD, chronickidney disease, hypertension, cardiovascular diseases such asasymptomatic left ventricular dysfunction, chronic congestive heartfailure and atherosclerosis. Renalase can also be used as a diagnosticmarker for acute renal failure (i.e. acute tubular necrosis, or ATN),and the like.

The method comprises obtaining a biological sample from the mammal andcomparing the level of renalase (expression, amount, activity) in thesample with the level of renalase in a sample from a person who is notafflicted with a renal disease. A lower level of renalase in the samplefrom the patient compared with the level of renalase in the sampleobtained from a person not afflicted with ESRD, ANT, or hypertension isan indication that the patient is afflicted with ESRD, ANT, orhypertension.

The invention includes a method of assessing the effectiveness of atreatment for ESRD in a mammal. The method comprises assessing the levelof renalase expression, amount, and/or activity, before, during andafter a specified course of treatment for ESRD since ESRD is associatedwith decreased renalase expression. This is because, as statedpreviously elsewhere and demonstrated by the data disclosed herein,renalase expression, amount and/or activity is associated with orincreased catecholamine circulation which is feature of certain diseasestates (e.g., ESRD and hypertension). Thus, assessing the effect of acourse of treatment upon renalase expression/amount/activity indicatesthe efficacy of the treatment such that a increased level of renalaseexpression, amount, or activity indicates that the treatment method issuccessful.

Without further description, one of ordinary skill in the art can, usingthe preceding description and the following illustrative examples, makeand utilize the compounds of the present invention and practice theclaimed methods. The following working examples therefore, specificallypoint out the preferred embodiments of the present invention, and arenot to be construed as limiting in any way.

EXAMPLES Overview

The experiments presented in this example may be summarized as follows.First, renalase was identified. To isolate this novel kidney-secretedproteins, the existing public databases, specifically the Mammalian GeneCollection Project (MGC) was utilized. A total of 114 candidate genesencoding novel secretory proteins was identified based on the followingcriteria: (i) candidate genes encode novel proteins, with less than 20%similarity/identity to existing proteins in the data base; (ii) putativeproteins are predicted to harbor the signal peptide sequence (using 2different methods of predicting signal peptide sequence); (iii) putativeproteins do not contain transmembane domains (since some membraneproteins, such as type I membrane proteins, also harbor a signal peptidesequence).

This strategy offers several distinct advantages: analysis is restrictedto novel genes only; it bypasses the cumbersome cloning process,trimming months or even years off of the search for an interesting gene;it allows immediate verification of gene expression in tissues, and ofbiochemical and function studies. Indeed, using this algorithm, oneclone, MGC12474, was found to be highly expressed in the kidney (seebelow). Thus, this clone was chosen for further study.

It was found that MGC12474 encodes a protein with monoamine oxidase(MAO) activity. MAO is a flavin-adenosine-dinucleotide (FAD)-containingenzyme, which converts biogenic monoamines to their correspondingaldehydes. The enzymatic reaction (Massey et al., 2000) catalyzes theoxidation of monoamines via an oxidative cleavage of the α-CH bond ofthe substrate to form an imine product with the concomitant reduction ofthe flavin cofactor. The imine product is then hydrolyzed to thecorresponding aldehyde and ammonia. The reduced flavin coenzyme reactswith oxygen to form hydrogen peroxide and the oxidized form of theflavin to complete the catalytic cycle.

Rabbit anti-renalase polyclonal antibody was also raised using asynthetic peptide derived from amino acid position 226-238 that isidentical between human and mouse. Western blot study showed that thisantibody recognized the same 37 Kd renalase-HA fusion protein as anti-HAantibody.

Moreover, it was found that renalase is secreted to the blood (see belowfor detail) in human, further demonstrating the nature of renalase beinga secretory protein.

In order to facilitate the detection of the protein product, a HA tag atthe C-terminus of renalase was engineered. Western blotting with bothanti-HA and anti-renalase antibodies revealed a 37 Kd protein in theculture medium of kidney-derived HEK293 cells, indicating that renalasehas a functional N-terminal signal sequence, and is secreted in the cellculture model used in these studies.

Furthermore, human blood was examined by western blotting using arenalase specific polyclonal antibody. FIG. 3 b indicates that renalaseis easily detectable in blood. To determine if the kidney is the majorsource of circulating renalase, we tested if blood levels were reducedin patients with severe kidney disease and decrease renal function. Asshown in FIG. 3 b, renalase was virtually undetectable in the blood ofpatients with ESRD receiving hemodialysis.

The experiments are described in more detail below.

Example 1 Identification and Analysis of the Gene Encoding RenalaseMaterial and Methods

Bioinformatics Analysis of MGC Database

All 12,563 distinct human full-ORF cDNAs available from Mammalian GeneCollection Project MGC as of the date of this experiment, were subjectedto 3 rounds of sequential screening. The initial analysis of MGC wasconducted on Dec. 24, 2001. First, genes without a GenBank Definitionwere chosen for more detailed analysis. Second, the predicted amino acidsequences encoded by genes selected in round 1 were analyzed using BLAST(http://www.ncbi.nlm.nih.gov/BLAST) and those encoding proteins withless than 20% identity with known proteins were chosen. Third, thepresence of putative signal sequences was assessed using SignalP V2.0(www.cbs.dtu.dk/services/SignalP-2.01) and SOSUI signal Beta_Version(http://sosui.proteome.bio.tuat.ac.jp). Novel proteins with signalpeptide sequence predicted by both methods were then subjected to domainsearch using Pfam. The cDNA clone encoding clones of interest werepurchased from ATCC, sequenced on both strands (Yale University, KeckFoundation Biotechnology Resource Laboratory) and analyzed using BLAST.

MGC

The MGC project is a new effort by the NIH to generate full-length cDNAresources to facilitate the functional studies of human genes. Thisproject provides publicly accessible resources to the worldwide researchcommunity. The MGC project entails the production of libraries,sequencing, and database and repository development, as well as thesupport of library construction, sequencing, and analytic technologiesaimed at obtaining a full set of human and other mammalian full-length(open reading frame) sequences and clones of expressed genes (Rozanskiet al., 1999; Tendera et al., 2001). Most importantly, MGC hasestablished robust informatics tools to ensure that the selected clonespotentially encode complete sequences. MGC produced 12,563 distincthuman full-ORF cDNAs as of the date of this experiment, about 20% ofwhich are novel genes (novel genes are defined as less than 20%similarity/identity to known proteins).

DNA Sequence

MGC12474 cDNA clone encoding renalase was purchased from ATCC and wassequenced on both strands (Yale University Keck Foundation BiotechnologyResource Laboratory) and analyzed for DNA sequence identity/similarityto the published sequence using BLASTN at the National Center forBiotechnology Information website (www.ncbi.nlm.nih.gov/BLAST/).

Northern Blot Analysis

Human multiple tissue Northern blot was obtained from CLONTECH andhybridized with the renalase cDNA labeled with [α-³²P]dCTP.Hybridizations were carried out in Rapid-hyb buffer (Amersham PharmaciaBiotech) containing labeled probe (˜2×10⁶ cpm/ml) at 62-68° C. for 1-2 hor overnight. The blot was washed under stringent conditions and exposedto Kodak XAR films. Glyceraldehyde-3-phosphate dehydrogenase cDNA wasused as probe for the internal control for equal RNA loading.

Statistical Analysis

Standard paired Student's t-tests were used for comparisons between twogroups. Standard unpaired Student's t-tests were used for groupcomparisons at equivalent periods. All data are means±SE, and P<0.05 wasaccepted as a statistically significant difference.

Results

In order to isolate novel proteins with import biological roles, theexisting public databases, specifically the Mammalian Gene CollectionProject (MGC) were screened (Strausberg et al., 1999), using analgorithm designed to select new proteins that are likely to be secretedby the kidney. The MGC project is a new effort by the NIH to generatefull-length cDNA resources in human and mouse to facilitate thefunctional study of the gene products (Strausberg et al., 1999;http://mgc.nci,nih.gov). To select candidate genes encoding secretedproteins, all the clones published by the MGC were studied. MGC produced12,563 distinct human full-ORF cDNAs as of the date of theseexperiments, about 20% of which are novel genes (novel genes are definedas less than 20% similarity/identity to known proteins). By the time theMGC data analysis was completed on Aug. 1, 2003, a total of 114candidate genes encoding novel secretory proteins were identified basedon the following criteria: (i) candidate genes encode novel proteins,with less than 20% similarity/identity to existing proteins in the database (ii) putative proteins are predicted to harbor the signal peptidesequence (using 2 different methods of predicting signal peptidesequence); (iii) putative proteins do not contain transmembane domains(since some of membrane proteins, such as type I membrane proteins, alsoharbor a signal peptide sequence). Tissue expression of each candidategene was assessed by Northern blot analysis and revealed robust andpreferential expression of one of these clones (MGC12474, GenBankaccession # BC005364) in human kidney (FIG. 1A). MGC12474 has 1,474nucleotides, and its longest open reading frame (nucleotides 22-1047)encodes a novel protein with 342 amino acids with a calculated molecularmass of 37.8 kDa (FIG. 2A). The human gene, which is named renalase, has7 exons spanning approximately 311,000 bp and resides on chromosome 10at q23.33 (FIG. 2B). Analysis, using MotifScan, revealed a signalpeptide at the N terminus, a FAD binding site (amino acid 4-35), and anamine oxidase domain at amino acids 11-339 (FIG. 1B). Renalase has 13.2%amino acid identity with monoamine oxidase A (MAO-A) (FIG. 1C) and has aweak similarity to MAO-B (FIG. 2C).

Example 2 Renalase is Secreted by the Kidney Construction of GeneExpression Cassettes (TAP Fragments)

We used a PCR-based approach that after two sequential PCR reactions,the 5′-CMV promoter and 3′-SV40pA were added to the 5′- and 3′-end ofeach candidate clone respectively. The detailed methodology is describedby Liang et al. In brief, this method is comprised of two sequential PCRsteps. The first step is carried out using primers (0.4 μg each)containing universal TAP ends and sequences specific to the target gene.The 5′ universal end sequences are complementary to the DNA fragmentcontaining the CMV immediate early gene promoter and are used in thesecond PCR step to attach the CMV promoter to the amplified gene. The 3′universal end overlaps with a DNA fragment that contains the SV40 earlygene transcription terminator and is also used in the second-step PCR toattach the transcription terminator sequence to the amplified gene. Togenerate TAP fragment containing renalase, the 5′- and 3′-primers usedfor the first step PCR are as follows:5′-oligo=5′-TGCAGGCACCGTCGTCGACTTAACAatgcgaccccagggccccgccg (SEQ ID NO:6, upper case is the 5′-TAP universal sequence which is used as ananchor for second step PCR, lower case is the clone #2-specific sequencestarting at the ATG initiation site);3′-oligo=5′-CATCAATGTATCTTATCATGTCTGATCAACCAGCTACCCATACGATGTTCCAGATTACGCTttttggtagttcttcaataag (SEQ ID NO: 7, the first 25bases are 3′-TAP universal sequence which is used as an anchor forsecond step PCR, underlined sequence encode HA followed by a stop codonTGA, lower case is the clone renalase-specific sequence starting at3′-end minus the stop codon). The 5′- and 3′-primers used for the secondstep PCR are provided by the manufacturer (Gene Therapy Systems, Inc.San Diego. CA).

Gene Delivery and Expression

In vitro transfection was carried out using GenePORTER (Gene TherapySystems, Inc. San Diego. CA) following the procedures recommended by themanufacturer. We consistently obtained 40-60% transfection efficiency asassessed using a Green Florescence Protein TAP fragment as control.

In Vitro Translation

Renalase cDNA was cloned into pDNR-LIB which contains a T7 before the5′-end of the insert. Renlase mRNA was transcribed and subsequentlytranslated using the Single Tube Protein® System 3 (Novagen, CA). ALuciferase T7 cDNA was used as a positive control. In vitrotranscription-translation was carried out with 1 μg of plasmid DNA in 50μl of reaction mixture supplemented with 50 μCi of [³⁵S] methionine(Amersham Pharmacia Biotech, NJ) according to manufacturer'sinstruction. 10 μl of the products were separated by 10%SDS-polyacrylamide gel electrophoresis, followed by autoradiography andWestern blotting.

Gene Delivery and Expression

In vitro transfection was carried out using GenePORTER (Gene TherapySystems, Inc.) following the procedures recommended by the manufacturer.We consistently obtained 40-60% transfection efficiency using the GreenFlorescence Protein TAP fragment as control. To test whether renalase isa secreted protein, we employed a PCR-based approach to generatetranscriptionally active PCR (TAP) fragments that were used directly inin vivo expression experiments (Liang et al., 2002).

Renalase Antibody Generation.

The rabbit anti-renalase polyclonal antibody was generated byProteintech Group, Chicago) using recombinant GST-renalase fusionprotein as antigen. Ten weeks later, rabbits were boosted with theGST-renalase fusion protein. 6-8 weeks after the second injection ofantigen, rabbits were bled and the anti-renalase antibody wasaffinity-purified.

Western Blot Analysis.

HEK 239 cells were grown in Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum, 2 mM L-glutamine, andantibiotics. Cells were grown in six-well plates to 60-80% confluenceand then transfected with TAP fragments containing renalase-HA fusionprotein using Geneporter according to manufacturer's instruction. 48-72h after transfection, the proteins from cell lysate were separated on a10% SDS-polyacrylamide gel, transferred onto nitrocellulose membrane,and immunoblotted with an anti-HA monoclonal antibody (Roche Chemicals).

Immunocomplexes were detected with a secondary antibody conjugated tohorseradish peroxidase (Pierce) and visualized with SuperSignal WestPico Luminol/enhancer solution (Pierce). To examine the secretoryproperties of the proteins encoded by the candidate genes, the culturemedia were subjected to Western blot analysis in parallel to the celllysate. To examine the human plasma renalase, 10 microliters of plasmawere analyzed by Western blot using a renalase-specific antibody.

In Situ Hybridization

In situ hybridization was performed as described previously. In brief, a426-bp DNA fragment from MGC clone # 12474 was isolated by restrictionenzyme HindIII and PstI digestion. It was then subcloned into thepBluescript® II KS(+) vector and in vitro transcribed into DIG-labeledRNA probes with DIG-labeling kit (Roche Biochemicals). The anti-sensewas used to detect renalase, while the sense strand was used as control.These probes were used to hybridize with the sections cut from the heartand kidney tissues embedded in paraffin. The heart and kidney tissues ofhuman adult were obtained from autopsy cases with the consent of familymembers and the approval of the university clinical research ethicscommittee. Tissues were fixed overnight with 4% parafomaldehyde inphosphate-buffered saline (PBS). The postmortem delay was 7 hours. Thetissues were dehydrated through grade ethanol, cleared with xylene, andembedded in Parafilm, and 5-μm-thick sections were prepared.

After dewaxing and hydration, the sections were digested with ProteinaseK (20 μg/ml) at 37° C. for 10 min. They were then post-fixed with 4%paraformaldehyde in PBS for 10 min. Hybridization was performed at 50°C. in a hybridization buffer containing 4×SSC, 10% dextran sulfate,1×Denhardt's solution, 5 mM EDTA, 0.1% CHAPS, 50% deionized formamide,200 μg/ml salmon sperm DNA, and 200 ng/ml DIG-labeled probe. The slideswere washed four times for 15 min each in 2×SSC and then twice for 15min each in 0.2×SSC/0.1% SDS at 50° C. Colorimetric detections wereperformed using an anti-DIG antibody conjugated to alkaline phosphatasefollowed by incubation with NBT/BCIP color substrates using adigoxygenin-nucleic acid detection kit (Roche, Germany). For humanrenalase, 5 min was needed for the color development in NBT/BCIP colorsubstrate solution.

Results

To determine the tissue distribution of renalase mRNA, we performedNorthern blotting on panels of human tissues. The results depicted inFIG. 1A show that renalase mRNA is highly expressed in kidney with lowerlevels in several other tissue types. In situ hybridization studies werecarried out to determine the spatial distribution of renalase mRNA invarious human tissues (FIG. 3). A specific signal was detected in renalglomeruli, proximal tubules (FIG. 3A, left panel), and in cardiacmyocytes (FIG. 3B, left panel). This distribution was confirmed byimmunocytochemical experiments, as evidenced by the detection ofrenalase protein in renal glomeruli, proximal tubules (FIG. 3C, leftpanel), and in cardiac myocytes (FIG. 3D, left panels). These resultsindicate that high level renalase mRNA expression is tissue-specific,suggesting that it may have functions specific for cells found inkidney, skeletal muscles, heart and liver.

To test whether the candidate genes encode secreted proteins, weemployed a PCR-based approach to generate transcriptionally active PCR(TAP) fragments that are used directly in in vivo expression experiments(6). In order to facilitate the detection of protein product, we alsoengineered a HA tag at the C-terminus of renalase (we avoided theN-terminus to preserve the integrity of the signal peptide). As shown inFIG. 4 a, we fused the 5′-CMV promoter and 3′-SV40pA to the renalase-HA,we transfected the TAP fragment into HEK293 cells. Western blotting withanti-HA antibody revealed an expressed protein product of expected 37 Kdin the culture medium (FIG. 4 b), consistent with the presence of aputative N-terminal signal sequence.

Unlike MAO-A, MAO-B and PAO that are either membrane-bound or confinedto intracellular compartments, renalase is secreted into the blood,where it is detectable by western blotting. Amine oxidase activity hasbeen measured in human plasma, and is believed to be mediated byvascular adhesion protein 1 (VAP-1), a copper-containing semicarbizidesensitive amine oxidase that is secreted by smooth muscle, adipocytesand endothelial cells (Salmi et al., 2001). VAP-1's substratespecificity and inhibitor profile are very different that of renalase.It metabolizes benzylamine and methylamine, and is inhibited bysemicarbizide and hydroxylamine. Therefore, renalase is the only knownamine oxidase that is secreted into circulation and that metabolizescirculating catecholamines.

Renalase expression appears limited to the kidney, heart, skeletalmuscle and small intestine. The kidney exhibits the highest expressionlevel and appears to be responsible for the bulk of circulatingrenalase. Indeed, renalase levels are dramatically reduced in patientswith end-stage renal disease (ESRD) who are undergoing dialysis. Thepossibility cannot be excluded that the metabolic abnormalitiesassociated with severe renal failure could decrease renalase secretionby the heart and skeletal muscles. Nonetheless, it is likely that thekidney is an important contributor to circulating renalase pool.

Interestingly, recent studies have shown that plasma dopamine andnorepinephrine levels and sympathetic tone are consistently increased inpatients with ESRD (Joles et al., 2004; Zoccali et al., 2002; Hausberget al., 2002). A recent study has also found that sudden emotionalstress may increase sympathetic stimulation leading to myocardialstunning (Wittstein et al., (2005) Neurohumoral features of myocardialstunning due to sudden emotional stress, New England Journal ofMedicine. 352, 539-548). Heightened sympathetic tone may contribute tothe pathogenesis of cardiovascular complications such as hypertension,left ventricular hypertrophy and dysfunction. These disturbances arelikely contributors to the high mortality rate observed in patients withESRD. Thus, it is possible that low renalase blood levels lead to theheightened sympathetic tone observed in ESRD patients, and that renalaseadministration may decrease the incidence of cardiovascular complicationand improve survival.

Example 3 Renalase Degrades Catecholamines In Vitro and RegulatesCardiac Function and Systemic Blood Pressure In Vivo In VitroTranscription/Translation

MGC12474 clone was cloned into pDNR-LIB which contains a T7 before the5′-end of the insert. renalase mRNA was transcribed and subsequentlytranslated using the Single Tube Protein® System 3 (Novagen, Cat #70192). A Luciferase T7 cDNA was used as a positive control. In vitrotranscription-translation was carried out with 1 μg of plasmid DNA in 50μl of reaction mixture supplemented with 50 μCi of [³⁵S] methionine(Amersham Pharmacia Biotech) according to manufacturer's instruction. 10μl of the products were separated by 10% SDS-polyacrylamide gelelectrophoresis, followed by autoradiography and Western blotting.

Generation of GST-Renalase Recombinant Protein

The renalase coding region (nt 24-1052) is amplified with sense primer5′-TTTT GGA TCC ATG GCG CAG GTG CTG ATC GTG (SEQ ID NO: 8) andantisense: 3′-TTTT GAA TTC CTA AAT ATA ATT CTT TAA AGC (SEQ ID NO: 9).The PCR fragment, after digesting with Bam HI and Eco RI, was clonedinto the pGEX-4T (Promega) in frame with GST tag (26 kDa) at theN-terminus. After verifying the correct cloning by DNA sequencing, therecombinant GST-renalase plasmid was transformed into E. Coli BL21 forrecombinant fusion production. After transformation, 6 liter ofbacterial culture was grown at 37° C. at 220 rpm for 16 hours with IPTG(0.5 mM) added in the last 3.5 hours of culture. 10 μM of FAD was alsoadded at the time of IPTG induction. The presence of renalase wasconfirmed by Western Blotting with 40 μg protein of total bacteriumlysates.

Renalase proteins can be purified directly from bacterial lysates with aone-step method using GSTrap column. The binding buffer contains PBS pH7.0 (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH2 PO₄, pH 7.0.The elution buffer. 50 mM Tris™-HCl and 10 mM reduced glutathione, pH8.0. For sample preparation, 10 g E. coli sample was centrifuged andresuspened in binding buffer on ice. The sample was sonicated to releasethe GST-renalase fusion protein. The procedure of column purification isas follows:

-   -   1) Equilibrate the column with 5 column volumes of binding        buffer,    -   2) Apply the sample use a flow rate of 0.2 ml/min,    -   3) Wash with 5-10 column volumes of binding buffer or until no        material appears in the effluent    -   4) Elute with 5-10 column volumes of elution buffer.

One of the most important parameters affecting the binding of GST fusionproteins or other glutathione binding proteins to GSTrap is the flowrate. Due to the relatively slow binding kinetics between GST andglutathione, it is important to keep the flow rate low during sampleapplication for maximum binding capacity. Volumes and times used forelution may vary among different batch proteins. Additional elutionswith higher concentrations of glutathione may be required. Flow-through,wash and eluted material from the column should be monitored for GSTfusion proteins using SDS-PAGE in combination with Western Blot ifnecessary.

GST-Renalase Denaturation

Renalase protein denaturation by urea has been a subject of intensestudies and controversy. The study of renalase denaturation is carriedout as follows:

1. Prepare each of the two protein samples to be compared in 50 μl ofsample buffer containing 50 mM Tris-HCl (pH 8) at a final proteinconcentration of approximately 1 mg/ml.

2. Denature the proteins by adding 50 μl of sample (from step 1) to 18mg solid urea for a final urea concentration of 6 M.

3. Reduce the proteins by adding 5 μl of freshly-prepared 100 mM DTT(prepared in High-grade H₂O) and mix by vortexing. Incubate thesolutions at room temperature for 1 hour.

GST-Renalase Refolding

Efficient refolding process of denatured renalase, produced and purifiedto homogeneity, was denatured with 8M urea at neutral pH and rapidlydiluted using various buffers. Rapid dilution with neutral pH buffersyielded low protein recovery. Reduction of protein concentration in therefolding solution did not improve protein recovery. Rapid dilution withalkaline buffers also yielded low protein recovery. However, dilutionwith mildly acidic buffers showed quantitative protein recovery withpartial enzymatic activity, indicating that recovered protein was stillarrested in the partially refolded state. Therefore, we furtherinvestigated the efficient refolding procedures of partially refoldedrenalase formed in the acidic buffers at low temperature (4° C.).Renalase enzymatic activity remained constant at pH 4. The same pHtitration with incubation shorter than 12 h yielded less enzymaticactivity. Refolding trials performed at room temperature led toaggregation, with almost half of the activity yield obtained at 4degrees C. We conclude that rapid dilution of urea denatured Renalaseunder acidic pH at low temperature results in specific conformationsthat can then be converted to the native state by titration tophysiological pH. The process is carried out as follows:

1. Purify renalase protein as inclusion bodies and solublize in neutralbuffered 8M urea. Supplement the buffer with DTT as required by theprotein.

2. Adjust the protein concentration to 0.1 mg/ml.

3. Dialysed 1000 μl of each protein against dialysis buffers as showingin order.

4. Slowly add 1000 μl of the protein solution to each dialysis tubewhile mixing the solution gently.

5. Dialysis at 4° C. for 12 hour according the buffer sequence.

6. Collect the Renalase by microfuge for 5 min.

7. Carefully pipette the liquid into a clean tube. This should containrefolded, soluble protein.

8. Assess successful refolding as follows:

It is best to perform a functional assay to determine if any activeprotein is present. Misfolded or aggregated protein will have adifferent activity reading than the correctly folded protein. Successfulrefolding is achieved when >30% of the input protein or activity isrecovered in the soluble fraction.

Buffers used in the experiment include the following:

Buffer 1: 50 mM MES pH 5.5, 10 mM NaCl, 0.4 mM KCl, 2 mM MgCl2, 2 mMCaCl2, 0.75 M Guanidine HCl, 0.5% Triton X-100, 1 mM DTT, 0.1 mM FAD

Buffer 2: 50 mM MES pH 5.5, 10 mM NaCl, 0.4 mM KCl, 2 mM MgCl2, 2 mMCaCl2, 0.5 M arginine, 0.05% polyethylene glycol 3,550, 1 mM GSH, 0.1 mMGSSHBuffer 3: 50 mM MES pH 5.5, 10 mM NaCl, 0.4 mM KCl, 1 mM EDTA, 0.4 Msucrose, 0.75 M Guanidine HCl, 0.5% Triton X-100, 0.05% polyethyleneglycol 3,550, 1 mM DTTBuffer 4: 50 mM MES pH 5.5, 200 mM NaCl, 10 mM KCl, 2 mM MgCl2, 2 mMCaCl2, 0.5 M arginine, 0.5% Triton X-100, 1 mM GSH, 0.1 mM GSSHBuffer 5: 50 mM MES pH 5.5, 200 mM NaCl, 10 mM KCl, 1 mM EDTA, 0.4 Msucrose, 0.75 M Guanidine HC, 1 mM DTTBuffer 6: 50 mM MES pH 5.5, 200 mM NaCl, 10 mM KCl, 1 mM EDTA, 0.5 Marginine, 0.4 M sucrose, 0.5% Triton X-100, 0.05% polyethylene glycol3,550, 1 mM GSH, 0.1 mM GSSH.

Amine Oxidase Assay

Enzyme assay of renalase was carried out using an Amplex Red MonoamineOxidase Assay Kit (Molecular Probes, Cat # A-12214) that providesone-step fluorometric method for the continuous measurement of amineoxidase activity using a fluorescence microplate reader. The assay isbased on the detection of H₂O₂ in a horseradish peroxidase-coupledreaction using 10-acetyl-3,7-dihydroxy-phenoxazine (Amplex Red reagent),a highly sensitive and stable probe for H₂O₂. Experiments were carriedout according to manufacturer's instruction with a final substrateconcentration of 2 mM.

Results

Structural analysis revealed that renalase contains an amino oxidasedomain, suggesting it may play a role in amine oxidation. Therefore, wetested whether it had oxidase activity using a battery of amines assubstrates. Renalase fusion protein in E. Coli was generated by cloningrenalase cDNA into a Glutathione-S-Transferase (GST)-1-containing pGEXexpression vector using the GST Gene Fusion System. As shown in FIG. 4a, renalase specifically metabolizes cathecholamines with the followingrank order potency: dopamine>epinephrine>norepinephrine. Its enzymaticactivity was unaffected by inhibitors of the FAD-containing amineoxidases, MAO-A and MAO-B (FIG. 4 b).

The GST-renalase fusion protein was purified to homogeneity using aGlutathione Sepharose column (FIG. 5 a). The purified fusion protein hasa MW of ˜64 KD, in agreement with the MW of GST tag (26 KD) plus thepredicted MW of renalase (37.8 KD). Since renalase is highly expressedin the kidney and is a secretory protein, it is conceivable thatrenalase is present in the circulation and that individuals with ESRDhave much reduced level of renalase. When plasma samples are analyzedwith the renalase-specific antibody by Western blot (FIG. 5B), we foundthe levels of renalase in patients with ESRD on hemodialysis arevirtually undetectable, whereas the normal individuals have acirculating renalase concentration of about 7 mg/l.

We subsequently tested whether renalase has an oxidase activity using abattery of amines as substrates. As shown in FIG. 6, GST-renalase fusionprotein has significant oxidase activity when dopamine, norepinephrineand epinephrine (2 mM) were used as the substrate. Thus, it can beconcluded that renalase is novel protein that metabolizes dopamine,norepinephrine and epinephrine.

Example 4 Renalase Regulates Systemic Blood Pressure HemodynamicsMeasurements

Sprague Dawley rats (150-250 g) were anesthetized with inactin (100mg/kg). A catheter (PE-240) was placed in the trachea for airwayprotection and in the left jugular vein (PE-50) for intravenous infusionof a maintenance fluid solution consisting of normal saline with 6.25%bovine albumin, at a rate of 1.5 ml per 100 g body wt per hour. Coretemperature was monitored through a rectal thermometer and bodytemperature was maintained at 37° C. using a heating pad. Arterialpressure and heart rate were continuously monitored through a PE-50catheter inserted in the left carotid artery and connected to a pressuretransducer (ADInstruments, CO, USA). Hemodynamic recordings weredigitized, stored and analyzed using a PowerLab/8SP data acquisitionsystem (ADIntruments). The rats were allowed 1 h to recover aftercompletion of the surgical procedure, and the subsequent 30 minutesserved as a control period. The experimental group then received a bolusintravenous injection of 0.5 mg of recombinant renalase in 0.5 ml PBS.The control group was injected with either 0.5 mg BSA or 0.5 mgrecombinant glutathione transferase in 0.5 ml of PBS. Blood pressure andheart rate were continuously measured and recorded.

Blood pressure is a function of cardiac output and peripheral vascularresistance, and is regulated by the sympathetic nervous system.Circulating catecholamines control heart rate, myocardial contractility,and the tone of resistance vessels. Since renalase circulates in bloodand degrades catecholamines, we examined its in vivo effect onhemodynamic parameters. Within 30 seconds of a single bolus intravenousinjection of recombinant renalase, systolic, diastolic and mean arterialpressure decreased by 23.5±1.3, 32.6±2.9 and 28.9±2.7% respectively,(n=4, p<0.001). As shown in FIG. 7A (upper panel), blood pressurerecovered within 2 minutes, and thereafter progressively decreasedreaching a nadir (between 60-90 minutes) with systolic, diastolic andmean arterial pressures 16.5±1.5, 14.3±1.2, and 14.8±1.1% lower thanbaseline (n=4, p<0.01) (FIG. 7A, lower panel). Heart rate remainedunchanged initially, and decreased slightly (6.4%) by 60 min, (FIG. 7A,lower panel). In control studies, blood pressure and heart rate wereunaffected by either albumin or GST infusion (FIG. 7B). These dataindicate that the hypotensive action of renalase is most likely theresult of accelerated catecholamines degradation, which would preventthe expected rise in pulse rate (in response to hypotension), and woulddecrease myocardial contractility, and reduce vascular resistance.Alternatively, renalase could bind to a cognate receptor and modulatemyocardial contractility and vascular resistance directly.

TABLE 1 Control Renalase n p Mean Arterial Pressure (mmHg) 106.4 ± 4.7 65.3 ± 3.5 8 <0.0001 End systolic pressure (mmHg) 127.7 ± 8.1  92.7 ±2.7 5 <0.004 End diastolic pressure (mmHg) 11.3 ± 1.8  9.3 ± 1.5 5 NSHeart rate (beats/min) 342 ± 6  304 ± 9  5 <0.004 Cardiac Output(ml/min) 45.8 ± 2.8 33.2 ± 1.5 3 <0.03 dP/dt (mmHg/sec) 8604 ± 728 5235± 442 5 <0.001 Arterial elastance (mmHg/uL)  0.94 ± 0.09  0.8 ± 0.04 3NS Systemic vascular resistance (mmHg/L/min) 2323 ± 196 1966 ± 183 3 NS

Example 5 Animal Models Rat Remnant Kidney Model (RRKM)

A rat partial (5/6) nephrectomy or rat remnant kidney model can be usedas described (Wada, M et al; J Clin Invest 1997 Dec. 15;100(12):2977-83). Male rats (2-3 months old, weighing about 150-200 g)are subjected to unilateral nephrectomy (either left or right kidney)first. A 2.0 cm skin incision is made in the ventral midline, with itscranial terminus 1.0 cm caudal to the xyphoid process. A 2.0 cm muscleincision is made along the midline. The right kidney is isolated andcleared of surrounding fat and connective tissue to clearly view therenal artery and vein, and ureter as they enter the hilus of the kidney.Care is exercised to minimize disturbance of the adrenal gland. Aligature (3/0 silk) is placed around the renal artery, vein and ureter.These vessels are then cut proximal to the kidney and the kidney isremoved, taking care that the adrenal gland is not disturbed. The secondstep of the surgical procedure involves the removal of the 2/3 of theremaining kidney in 7-10 days after the first step. Plasma creatinine(Cr) and BUN levels rise dramatically due to the loss of renal mass andfunction. Over the next several weeks, plasma Cr and BUN levels ofsurviving animals decline somewhat toward normal values but remainelevated. Renal function then appears to remain relatively constant orstable for a period of variable duration. After this point, the animalsenter a period of chronic renal failure in which there is an essentiallylinear decline in renal function until death.

As surgical controls, age, weight-matched rats are subjected to a “sham”operation in which the kidneys are decapsulated but no renal tissue isremoved.

Intervention Model for Chronic Renal Failure

In this model, both nephrectomized and sham-operated rats are maintainedfor approximately 5-6 months after surgery. At this point, survivingnephrectomized animals will enter chronic renal failure phase.

Rats are divided into 8 groups with 15 rats in each group. Two groups ofnephrectomized rats are used as controls (Nx controls), with one ofthose groups receiving no treatment at all, while the other receivedinjections of only the vehicle buffer. In addition, two groups ofsham-operated rats were used as controls (sham controls), with one groupreceiving only the vehicle buffer, while the other received solublerenalase at 100 microgram/kg body weight. Four experimental groups ofnephrectomized rats are also employed, receiving renalase at 10, 100,500 microgram/kg body weight by SQ injection. Renalase treated andvehicle-only rats receive twice injection per day for 4-8 weeks.

Plasma BUN, Cr will be examined before and during the course of renalasetreatment in all groups. It is expected that renalase will offertreatment benefit to Nx rats (slow down chronic renal failureprogression). Histological studies will also be carried out the end ofrenalase treatment to examine the incidence of glomerular sclerosis,tubular collapse, interstitial sclerosis and microaneurysms.

Prophylactic Model for Chronic Renal Failure

In order to test the ability of renalase (renal therapeutic agent) toprevent, inhibit or delay the initiation of chronic renal failure, ratsare subjected to partial nephrectomies or sham-operated as describedabove. The rats are allowed to recover for approximately two weeks afterthe second step of surgery before initiation of renalase therapy. Atthis point, surviving animals are past the acute renal failure phase andhave not yet entered chronic renal failure. Rats are divided into twogroups of 12 rats. One group receives only vehicle buffer (Nx control)whereas the other receives renalase treatment at 100 mircrogram/kg bodyweight given SQ twice per day. Administration of renalase or vehiclecontinued for a period of approximately 8-9 weeks.

Plasma BUN, Cr will be examined before and during the course of renalaseinjection in all groups. It is expected that renalase will prevent,inhibit or delay the initiation of chronic renal failure. Histologicalstudies will also be carried out the end of renalase treatment toexamine the incidence of glomerular sclerosis, tubular collapse,interstitial sclerosis and microaneurysms.

Hypertensive Model

The spontaneously hypertensive outbred rats (SHR) is generally used forstudies in essential hypertension and cardiovascular research. Males,twelve weeks of age or older, dependably exhibit average systolic bloodpressures greater than 200 mmHg. The anti-hypertensive effect ofrenalase can be tested in SHR.

SHR Rats are divided into two groups of 12 rats. One group receives onlyvehicle buffer whereas the other receives renalase treatment at 100mircrogram/kg body weight given SQ twice per day. Administration ofrenalase or vehicle continued for a period of approximately 2-6 weeks.Rat blood pressure will be examined everyday by implantable telemetrydevice.

Congestive Heart Failure Model

Congestive heart failure (CHF) models are well described in theliteratures, including large and small animals. One can use these modelsto test the prophylactic and therapeutic effect of renalase. Forexample, As described by Delehanty et al (Delehanty J M et al., Am J.Physiol. 1994 March; 266 (3 Pt 2): H930-5), one can use the rapidventricular pacing model to induce CHF in canines (i.e., dogs). Dogssubjected to pacing at 225 beats/min for 8 wk developed heart failure asevidenced by elevated left atrial pressure, depressed first derivativeof left ventricular pressure with respect to time, and depressed cardiacoutput compared with dogs paced at 100 beats/min for 8 wk. Fast-paceddogs also exhibited an elevated plasma NE and reduced myocardial NEcontent.

Other CHF animal models can be used as well. For example, maleSprague-Dawley (SD) rats are subjected to left coronary arterialligation as described previously (Greenen, D. L. et al., J. Appl.Physiol. 63:92-96 (1987); Buttrick, P. et al., Am. J. Physiol.260:11473-11479 (1991)) to induce myocardial infarction. The rats areanesthetized with sodium pentobarbital (60) mg/kg, ip), intubated viatracheotomy, and ventilated by a respirator. After a left-sidedthoracotomy, the left coronary artery is ligated approximately 2 mm fromits origin with a 7-0 silk suture. Sham animals undergo the sameprocedure except that the suture is passed under the coronary artery andthen removed. In 4-6 weeks after ligation myocardial infarction candevelop heart failure in rats. In clinical patients, myocardialinfarction or coronary artery disease is the most common cause of heartfailure. Congestive heart failure in this model reasonably mimicscongestive heart failure in most human patients.

Stroke (Cerebral Vascular Accident) Model

A “stroke” is a sudden loss of function caused by an abnormality in theblood supply to the brain. Stroke presents with different levels ofseverity ranging from “transient ischemic attack” or “TIA” (no permanentdisability), to “partial nonprogressing stroke”, to “complete stroke”(permanent, calamitous neurological deficit).

Stroke-prone spontaneously hypertensive (SHR-SP) rat are commonly usedfor stroke model. This experiment can be used to test for the possiblebeneficial effect(s) of renalase in stroke prevention/treatment. Male8-week old SHR-SP rats are divided in random order into 2 groups.Control rats are maintained on ordinary chow and water containing 1%NaCl as drinking solution, control group receive vehicle injection. Thetreatment group receives renalase injection SQ (100 microgram/kg, twicedaily) for 4-8 weeks. Systolic blood pressure will be measured bytail-cuff method in conscious animals, the stroke rate will be examinedusing magnetic resonance imaging (MRI), histopathology, andneurobehavioral testing in these two groups.

Arthrosclerosis Model

It is known that catecholamines cause vascular injury and, in thepresence of hyperlipidaemia, cause accelerated and aggravatedatherosclerosis (Kukreja R S et al, Atherosclerosis. (1981) 40 (3-4):291-8.). Therefore, renalase may prevent/treat atherosclerosis. Asdescribed by Kukreja et al (Kukreja R S et al, Atherosclerosis. 1981November-December; 40(3-4):291-8.), advanced aortic and coronaryatherosclerosis can be produced in rhesus monkeys by means of twoprocedures: (a) high fat and cholesterol feeding for 7 months, and (b)this diet coupled with daily i.v. injection of adrenaline (50micrograms/kg body weight). Monkeys subjected to procedure (b) willdevelop markedly advanced atherosclerosis in the form of fibrous plaquesin the aorta and coronary artery, while these lesions are expected to bemuch less frequent in the other group. The ratio of total to free serumcholesterol will be significantly increased and the aortic cholesterolcontent will be very high in monkeys subjected to both the atherogenicdiet and adrenaline injections. These models can be used to test theeffect of renalase on atherosclerosis prevention and treatment.

A second model for testing renalase involves apoE knockout mice (Zhang SH, Reddick R L, Piedrahita J A, Maeda N., (1992) Spontaneoushypercholesterolemia and arterial lesions in mice lacking apolipoproteinE, Science, 258, 468-471). The apoe knockout mouse was created by genetargeting in embryonic stem cells to disrupt the apoe gene. ApoE, aglycoprotein, is a structural component of very low density lipoprotein(VLDL) synthesized by the liver and intestinally synthesizedchylomicrons. It is also a constituent of a subclass of high densitylipoproteins (HDLs) involved in cholesterol transport activity amongcells. One of the most important roles of apoe is to mediate highaffinity binding of chylomicrons and VLDL particles that contain apoe tothe low density lipoprotein (LDL) receptor. This allows for the specificuptake of these particles by the liver which is necessary for transportpreventing the accumulation in plasma of cholesterol rich remnants. Thehomozygous inactivation of the apoE gene results in animals that aredevoid of apoE in their sera. The mice appear to develop normallyhowever they exhibit five times the normal serum plasma cholesterol andspontaneous atherosclerotic lesions. This is similar to a disease inpeople who have a variant form of the apoe gene that is defective inbinding to the LDL receptor and are at risk for early development ofatherosclerosis, and increased plasma triglyceride and cholesterollevels. The apoe knockout mice are widely used to as atherosclerosismodel to investigate intervention therapies that modify the atherogenicprocess and can be used herein for testing the effects of such therapiesusing renalase.

Example 6 Further Studies

In addition to the foregoing examples, studies have been planned orbeing conducted by the present inventors. One study involves clinicalassessment of the correlation of renalase levels with kidney function inabout 300 subjects. 70 people have been enrolled so far. Preliminaryresults will be available in about 6-7 weeks. In another study, theinventors will evaluate the effect of chronic renalase administration bysubcutaneous injection on the progression of chronic kidney disease andthe development of cardiovascular complications in a rat model ofchronic renal failure. In yet another study, the inventors will evaluatethe efficacy of intramuscular injections of a CMV-renalase vector in therat model.

Discussion

The renalase identified herein is the third monoamine oxidase in humanusing functional genomic database. Similar to MAO-A and MAO-B, renalasemetabolizes catecholamines such as dopamine (DA), norepinephrine (NA),and epinephrine (EP). Renalase has several important, unique featuresthat differentiate it from MAO-A and MAO-B. First, compared to MAO-A andMAO-B, renalase is predominantly expressed in the kidney, suggestingthat renalase has a unique role in catecholamine metabolism in theperiphery. It is believed that the metabolism of circulatory monoaminesare carried out by a host of intracellular enzymes (Eisenhofer et al.,2001), including MAO-A, MAO-B, catechol-O-methyltransferase (COMT) andsulfotransferase. Since these enzymes have intracellular locations, theprimary mechanism limiting the lifespan of circulatory catecholamine isuptake by active transport into cells, not metabolism by enzymes. Thisis consistent with the notion that the primary role of MAO-A and MAO-Blies in the metabolism of amines and in the regulation ofneurotransmitter levels and intracellular amine stores.

Second, unlike MAO-A and MAO-B that are located in the outer membrane ofmitochondrion, renalase is a secretory protein, suggesting catecholamineclearance can take place in the extracellular space, challenging thetraditional thinking that catecholamines have to be taken up by thecells in order to be catabolized. It is circulating in the blood whererenalase catabolizes serum catecholamine. It is possible that renalase“fine-tunes” the plasma catecholamine level from minute-to-minute.

Third, renalase is highly expressed in the kidney, suggesting the kidneyis involved in catecholamine degradation via the renalase catalyticpathway. The data provided herein is consistent with the hypothesis thatrenalase regulates catecholamine at the systemic level as well as atlocal kidney level. It is conceivable that renalase, in conjunction withMAO-A and MAO-B that catabolize intracellular amines, is an importantenzyme to oxidase extracellular catecholamine, and thus contributing tothe regulation of overall sympathetic tone.

Renalase is most abundant in the proximal tubules and it presents in thecirculation of normal individuals, suggesting that renalase protein inthe proximal tubules can be secreted via the basolateral membrane intothe circulation where it catabolized its substrate(s), and thus,regulating catecholamine homeostasis at a systemic level.

It is possible that renalase also exerts its biological function at thelumen of renal tubules, since renalase is small protein which can beeasily filtered to the lumen of nephron. In addition, renalase can bedirectly secreted via the apical membrane by the proximal tubules, whereit metabolize its substrate(s) that filtered through the glomeurli andgenerated de novo by the renal tubular cells such as dopamine (Wang etal., 2001). The significance of renalase in catecholamine metabolism atintra-lumen is to regulate intra-lumen catecholamine level, thusregulating salt and water re-absorption.

Recent studies have shown that plasma dopamine (Cuche et al., 1986;Prinseau et al., 1986) and norepinephrine (Zoccalie et al., 2002) isconsistently elevated in patients with ESRD. Those changes ofcatecholamine levels are important contributors to the pathogenesis ofcardiovascular diseases such as asymptomatic left ventriculardysfunction (Benedict et al., 1996), chronic congestive heart failure(Rouleau et al., 1994) and atherosclerosis (Rozanski et al., 1999). Theimportance of high sympathetic tone in cardiovascular complications isalso supported by intervention studies (Tendera et al., 2001). Inpatients with ESRD, increased circulating catecholamines might renderuremic patients susceptible to a series of cardiovascular complicationsranging from left ventricular hypertrophy to arrhythmia (Zoccali et al.,2002). The mechanism of elevated catecholamine in ESRD patients remainspoorly understood. Our discovery of renalase (which catabolizescatecholamines and is highly expressed in the kidney) may explain thepathogenesis of catecholamine derangement in those patients as they loserenal mass (similar to reduced erythropoetin secretion in ESRDpatients).

It is not surprising to see a dramatically reduced renalase in patientswith ESRD since renalase is mostly expressed in the kidney, an organthat ESRD patients have lost its function. The fact that renalasecatabolize catecholamine, coupled with elevated catecholamine level inESRD patients, strongly suggest renalase is a critical protein inmaintaining catecholamine homeostasis, and MAO-C is the missing factorthat contributes to catecholamine derangement leading to hypertension,cardiovascular diseases such as asymptomatic left ventriculardysfunction, chronic congestive heart failure and atherosclerosis.

The foregoing detailed description has been given for clearness ofunderstanding only and no unnecessary limitations should be understoodtherefrom as modifications will be obvious to those skilled in the art.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

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The disclosures of each and every patent, patent application, andpublication cited herein including but limited to the references listedimmediately below are hereby incorporated herein by reference in theirentirety.

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1-69. (canceled)
 70. A method for producing renalase protein capable ofmetabolizing catecholamines, comprising, synthesizing recombinantrenalase protein in a host cell in the presence of flavin adeninedinucleotide (FAD), and recovering the renalase protein.
 71. The methodof claim 70, wherein the renalase protein comprises an amino acidsequence having at least 80% sequence identity to SEQ ID NO:2.
 72. Themethod of claim 70, wherein the renalase protein comprises an amino acidsequence having at least 90% sequence identity to SEQ ID NO:2.
 73. Themethod of claim 70, wherein the renalase protein comprises an amino acidsequence having at least 95% sequence identity to SEQ ID NO:2.
 74. Themethod of claim 70, wherein the renalase protein comprises the aminoacid sequence of SEQ ID NO:2.
 75. The method of claim 70, wherein therenalase protein comprises an amino acid sequence encoded by SEQ ID NO:1or SEQ ID NO:3.
 76. The method of claim 70, wherein the catecholamine isdopamine, epinephrine, or norepinephrine.
 77. The method of claim 70,wherein the host cell is a bacterial, yeast, insect, or vertebrate hostcell.
 78. The method of claim 77, wherein the host cell is E. coli. 79.The method of claim 77, wherein the host cell is a yeast.
 80. The methodof claim 77, wherein the host cell is mammalian.
 81. The method of claim70, wherein FAD is added at the time of inducing protein synthesis. 82.The method of claim 70, wherein the renalase protein comprises apurification tag.
 83. The method of claim 82, wherein the purificationtag is glutathione-5-transferase (GST), maltose binding protein (MBP),influenza virus hemaglutinin (HA), HIS₆, or FLAG.
 84. The method ofclaim 70, wherein the renalase protein is recovered by affinitychromatography.
 85. The method of claim 70, wherein the renalase proteinis further denatured and refolded.
 86. The method of claim 85, whereinthe renalase protein is recovered in inclusion bodies, denatured withurea, and refolded.
 87. The renalase protein produced by the method ofclaim 70.